Human Receptor Proteins; Related Reagents and Methods

ABSTRACT

Nucleic acids encoding mammalian, e.g., primate or rodent receptors, purified receptor proteins and fragments thereof. Antibodies, both polyclonal and monoclonal, are also provided. Methods of using the compositions for both diagnostic and therapeutic utilities are provided.

This filing is a conversion of U.S. Provisional Patent Application60/077,329, filed Mar. 9, 1998, which is incorporated herein byreference, to a U.S. Utility Patent Application.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for affectingmammalian physiology, including morphogenesis or immune system function.In particular, it provides nucleic acids, proteins, and antibodies whichregulate development and/or the immune system. Diagnostic andtherapeutic uses of these materials are also disclosed.

BACKGROUND OF THE INVENTION

Recombinant DNA technology refers generally to techniques of integratinggenetic information from a donor source into vectors for subsequentprocessing, such as through introduction into a host, whereby thetransferred genetic information is copied and/or expressed in the newenvironment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host. See, e.g.,Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, (2ded.), vols. 1-3, CSH Press, NY.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the “immunenetwork”. Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of the immuneresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play critical roles in controllingthese cellular interactions. The interferons are generally considered tobe members of the cytokine family. Thus, there is considerable interestin the isolation, characterization, and mechanisms of action of cellmodulatory factors, an understanding of which will lead to significantadvancements in the diagnosis and therapy of numerous medicalabnormalities, e.g., immune system disorders.

Lymphokines apparently mediate cellular activities in a variety of ways.See, e.g., Paul (ed. 1996) Fundamental Immunology 3d ed., Raven Press,New York; and Thomson (ed. 1994) The Cytokine Handbook 2d ed., AcademicPress, San Diego. They have been shown to support the proliferation,growth, and/or differentiation of pluripotential hematopoietic stemcells into vast numbers of progenitors comprising diverse cellularlineages which make up a complex immune system. Proper and balancedinteractions between the cellular components are necessary for a healthyimmune response. The different cellular lineages often respond in adifferent manner when lymphokines are administered in conjunction withother agents.

Cell lineages especially important to the immune response include twoclasses of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types.

One means to modulate the effect of a cytokine upon binding to itsreceptor, and therefore potentially useful in treating inappropriateimmune responses, e.g., autoimmune, inflammation, sepsis, and cancersituations, is to inhibit the receptor signal transduction.Unfortunately, finding reagents capable of serving as an antagonist oragonist has been severely hampered by the failure to fully identify allof the components within the signaling systems. In order to characterizethe structural properties of a cytokine receptor in greater detail andto understand the mechanism of action at the molecular level, purifiedreceptor will be very useful. The receptors provided herein, bycomparison to other receptors or by combining structural components,will provide further understanding of signal transduction induced byligand binding.

The isolated receptor gene should provide means to generate aneconomical source of the receptor, allow expression of more receptors ona cell leading to increased assay sensitivity, promote characterizationof various receptor subtypes and variants, and allow correlation ofactivity with receptor structures. Moreover, fragments of the receptormay be useful as agonists or antagonists of ligand binding. See, e.g.,Harada, et al. (1992) J. Biol. Chem. 267:22752-22758. Often, there areat least two critical subunits in the functional receptor. See, e.g.,Gonda and D'Andrea (1997) Blood 89:355-369; Presky, et al. (1996) Proc.Nat'l Acad. Sci. USA 93:14002-14007; Drachman and Kaushansky (1995)Curr. Opin. Hematol. 2:22-28; Theze (1994) Eur. Cytokine Netw.5:353-368; and Lemmon and Schlessinger (1994) Trends Biochem. Sci.19:459-463.

From the foregoing, it is evident that the discovery and development ofnew soluble proteins and their receptors, including ones similar tolymphokines, should contribute to new therapies for a wide range ofdegenerative or abnormal conditions which directly or indirectly involvedevelopment, differentiation, or function, e.g., of the immune systemand/or hematopoietic cells. In particular, the discovery andunderstanding of novel receptors for lymphokine-like molecules whichenhance or potentiate the beneficial activities of other lymphokineswould be highly advantageous. The present invention provides newreceptors for ligands exhibiting similarity to cytokine likecompositions and related compounds, and methods for their use.

SUMMARY OF THE INVENTION

The present invention is directed to novel receptors related to cytokinereceptors, e.g., primate or rodent, cytokine receptor like molecularstructures, designated DNAX Interferon-like Receptor Subunits (DIRS),and their biological activities. In particular, it provides descriptionof two different subunits, designated DIRS1 and DIRS2. It includesnucleic acids coding for the polypeptides themselves and methods fortheir production and use. The nucleic acids of the invention arecharacterized, in part, by their homology to cloned complementary DNA(cDNA) sequences enclosed herein.

The present invention provides, in polypeptide embodiments: asubstantially pure or recombinant DIRS1 polypeptide comprising at leastthree distinct nonoverlapping segments of at least four amino acidsidentical to segments of SEQ ID NO: 2; a substantially pure orrecombinant DIRS1 polypeptide comprising at least two distinctnonoverlapping segments of at least five amino acids identical tosegments of SEQ ID NO: 2; a natural sequence DIRS1 comprising mature SEQID NO: 2; a fusion polypeptide comprising DIRS1 sequence; asubstantially pure or recombinant DIRS2 polypeptide comprising at leastthree distinct nonoverlapping segments of at least ten amino acidsidentical to segments of SEQ ID NO: 4; a substantially pure orrecombinant DIRS2 polypeptide comprising at least two distinctnonoverlapping segments of at least eleven amino acids identical tosegments of SEQ ID NO: 4; a natural sequence DIRS2 comprising SEQ ID NO:4; or a fusion polypeptide comprising DIRS2 sequence. Preferredembodiments include, e.g., the substantially pure or isolated antigenic:DIRS1 polypeptide, wherein the distinct nonoverlapping segments ofidentity: include one of at least eight amino acids; include one of atleast four amino acids and a second of at least five amino acids;include at least three segments of at least four, five, and six aminoacids, or include one of at least twelve amino acids; or DIRS2polypeptide, wherein the distinct nonoverlapping segments of identity:include one of at least thirteen amino acids; include one of at leasteleven amino acids and a second of at least thirteen amino acids;include at least three segments of at least ten, eleven, and twelveamino acids; or include one of at least twenty-five amino acids. Otherembodiments include compositions where: the DIRS1 polypeptide: comprisesa mature sequence of Table 1; is an unglycosylated form of DIRS1; isfrom a primate, such as a human; comprises at least seventeen aminoacids of SEQ ID NO: 2; exhibits at least four nonoverlapping segments ofat least seven amino acids of SEQ ID NO: 2; is a natural allelic variantof DIRS1; has a length at least about 30 amino acids; exhibits at leasttwo non-overlapping epitopes which are specific for a primate DIRS1; isglycosylated; has a molecular weight of at least 30 kD with naturalglycosylation; is a synthetic polypeptide; is attached to a solidsubstrate; is conjugated to another chemical moiety; is a 5-fold or lesssubstitution from natural sequence; or is a deletion or insertionvariant from a natural sequence; or the DIRS2 polypeptide: comprises amature sequence of Table 2; is an unglycosylated form of DIRS2; or isfrom a primate, such as a human; comprises at thirty-five amino acids ofSEQ ID NO: 4; exhibits at least four nonoverlapping segments of at leasttwelve amino acids of SEQ ID NO: 4; is a natural allelic variant ofDIRS2; has a length at least about 30 amino acids; exhibits at least twonon-overlapping epitopes which are specific for a primate DIRS2; isglycosylated; has a molecular weight of at least 30 kD with naturalglycosylation; is a synthetic polypeptide; is attached to a solidsubstrate; is conjugated to another chemical moiety; is a 5-fold or lesssubstitution from natural sequence; or is a deletion or insertionvariant from a natural sequence. Various combination compositionsinclude those comprising: a substantially pure DIRS1 and anotherInterferon Receptor family member; a substantially pure DIRS2 andanother Interferon Receptor family member; a sterile DIRS1 polypeptide;a sterile DIRS2 polypeptide; the DIRS1 polypeptide and a carrier,wherein the carrier is: an aqueous compound, including water, saline,and/or buffer; and/or formulated for oral, rectal, nasal, topical, orparenteral administration; or the DIRS2 polypeptide and a carrier,wherein the carrier is: an aqueous compound, including water, saline,and/or buffer; and/or formulated for oral, rectal, nasal, topical, orparenteral administration.

Fusion polypeptide embodiments include those comprising: mature proteinsequence of Table 1; mature protein sequence of Table 2; a detection orpurification tag, including a FLAG, His6, or Ig sequence; or sequence ofanother interferon receptor protein. Kit embodiments are provided, e.g.,a kit comprising such a polypeptide, and: a compartment comprising theprotein or polypeptide; or instructions for use or disposal of reagentsin the kit.

The invention also provides a binding compound comprising an antigenbinding site from an antibody, which specifically binds to a: naturalDIRS1 polypeptide, wherein: the binding compound is in a container; theDIRS1 polypeptide is from a human; the binding compound is an Fv, Fab,or Fab2 fragment; the binding compound is conjugated to another chemicalmoiety; or the antibody: is raised against a peptide sequence of amature polypeptide of Table 1; is raised against a mature DIRS1; israised to a purified human DIRS1; is immunoselected; is a polyclonalantibody; binds to a denatured DIRS1; exhibits a Kd to antigen of atleast 30 μM; is attached to a solid substrate, including a bead orplastic membrane; is in a sterile composition; or is detectably labeled,including a radioactive or fluorescent label; or a natural DIRS2polypeptide, wherein: the binding compound is in a container; the DIRS2protein is from a human; the binding compound is an Fv, Fab, or Fab2fragment; the binding compound is conjugated to another chemical moiety;or the antibody: is raised against a peptide sequence of a maturepolypeptide of Table 2; is raised against a mature DIRS2; is raised to apurified human DIRS2; is immunoselected; is a polyclonal antibody; bindsto a denatured DIRS2; exhibits a Kd to antigen of at least 30 μM; isattached to a solid substrate, including a bead or plastic membrane; isin a sterile composition; or is detectably labeled, including aradioactive or fluorescent label. Kit embodiments include, e.g., thosecomprising the binding compound, and: a compartment comprising thebinding compound; or instructions for use or disposal of reagents in thekit.

Various methods are provided, e.g., of producing an antigen:antibodycomplex, comprising contacting under appropriate conditions: a primateDIRS1 polypeptide with a described antibody; or a primate DIRS2polypeptide with a described antibody; thereby allowing the complex toform. In certain situations, the method is used wherein: the complex ispurified from other interferon receptors; the complex is purified fromother antibody; the contacting is with a sample comprising aninterferon; the contacting allows quantitative detection of the antigen;the contacting is with a sample comprising the antibody; or thecontacting allows quantitative detection of the antibody.

Other compositions comprise: a sterile binding compound as described, orthe described binding compound and a carrier, wherein the carrier is: anaqueous compound, including water, saline, and/or buffer; and/orformulated for oral, rectal, nasal, topical, or parenteraladministration.

Nucleic acid embodiments include, e.g., an isolated or recombinantnucleic acid encoding the: described DIRS1 polypeptide, wherein the:DIRS1 is from a human; or the nucleic acid: encodes an antigenic peptidesequence of Table 1; encodes a plurality of antigenic peptide sequencesof Table 1; exhibits identity over at least thirteen nucleotides to anatural cDNA encoding the segment; is an expression vector; furthercomprises an origin of replication; is from a natural source; comprisesa detectable label; comprises synthetic nucleotide sequence; is lessthan 6 kb, preferably less than 3 kb; is from a primate; comprises anatural full length coding sequence; is a hybridization probe for a geneencoding the DIRS1; or is a PCR primer, PCR product, or mutagenesisprimer; or the described DIRS2 polypeptide, wherein the: DIRS2 is from ahuman; or the nucleic acid: encodes an antigenic peptide sequence ofTable 2; encodes a plurality of antigenic peptide sequences of Table 2;exhibits identity over at least 30 nucleotides to a natural cDNAencoding the segment; is an expression vector; further comprises anorigin of replication; is from a natural source; comprises a detectablelabel; comprises synthetic nucleotide sequence; is less than 6 kb,preferably less than 3 kb; is from a primate; comprises a natural fulllength coding sequence; is a hybridization probe for a gene encoding theDIRS2; or is a PCR primer, PCR product, or mutagenesis primer.

The invention further provides a cell or tissue comprising the describedrecombinant nucleic acid. Certain embodiments include wherein the cellis: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeastcell; an insect cell; a mammalian cell; a mouse cell; a primate cell; ora human cell. Kits are also provided, e.g., the described nucleic acidand: a compartment comprising the nucleic acid; a compartment furthercomprising a primate DIRS1 polypeptide; a compartment further comprisinga primate DIRS2 polypeptide; or instructions for use or disposal ofreagents in the kit.

In other embodiments, the invention provides a nucleic acid which:hybridizes under wash conditions of 30 minutes at 30° C. and less than2M salt to the coding portion of SEQ ID NO: 1; hybridizes under washconditions of 30 minutes at 30° C. and less than 2M salt to the codingportion of SEQ ID NO: 3; exhibits identity over a stretch of at leastabout 30 nucleotides to a primate DIRS1 sequence; or exhibits identityover a stretch of at least about 30 nucleotides to a primate DIRS2sequence. Preferred embodiments include those nucleic acids wherein: thewash conditions are at 45° C. and/or 500 mM salt; or the stretch is atleast 55 nucleotides. Other embodiments include those nucleic acidswherein: the wash conditions are at 55° C. and/or 150 mM salt; or thestretch is at least 75 nucleotides.

The invention further provides a method of modulating physiology ordevelopment of a cell or tissue culture cells comprising contacting thecell with an agonist or antagonist of a mammalian DIRS1 or DIRS2. Themethod may involve where the cell is transformed with a nucleic acidencoding a DIRS1 or DIRS2 and another cytokine receptor subunit.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Outline I. General II.Activities

III. Nucleic acids

A. encoding fragments, sequence, probes

B. mutations, chimeras, fusions

C. making nucleic acids

D. vectors, cells comprising

IV. Proteins, Peptides

A. fragments, sequence, immunogens, antigens

B. muteins

C. agonists/antagonists, functional equivalents

D. making proteins

V. Making nucleic acids, proteins

A. synthetic

B. recombinant

C. natural sources

VI. Antibodies

A. polyclonals

B. monoclonal

C. fragments; Kd

D. anti-idiotypic antibodies

E. hybridoma cell lines

VII. Kits and Methods to quantify DIRS

A. ELISA

B. assay mRNA encoding

C. qualitative/quantitative

D. kits

VIII. Therapeutic compositions, methods

A. combination compositions

B. unit dose

C. administration

IX. Screening X. Ligands I. General

The present invention provides the amino acid sequences and DNAsequences of mammalian, herein primate, interferon receptor-like subunitmolecules, these ones designated DNAX Interferon Receptor family Subunit1 (DIRS1) and DNAX Interferon Receptor family Subunit 2, havingparticular defined properties, both structural and biological. VariouscDNAs encoding these molecules were obtained from primate, e.g., human,cDNA sequence libraries. Other primate or other mammalian counterpartswould also be desired. Descriptions, methods, and manipulations directedto DIRS1 may be applied, as appropriate, to DIRS2.

Some of the standard methods applicable are described or referenced,e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, etal. (1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3,CSH Press, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and periodic supplements)Current Protocols in Molecular Biology, Greene/Wiley, New York; each ofwhich is incorporated herein by reference.

A partial nucleotide (SEQ ID NO: 1) and corresponding amino acidsequence (SEQ ID NO: 2) of a human DIRS1 coding segment is shown inTable 1. Partial human DIRS2 sequence is provided (SEQ ID NO: 3 and 4).

TABLE 1 Nucleotide and amino acid sequences of DNAX IFN ReceptorSubunit like embodiments (DIRS1), originally designated HKAEF92.Primate, e.g., human embodiment (see SEQ ID NO: 1 and 2). Nucleotides567, 573, 1336, 1342, and 1369 are designated C, but may be A, C, G, orT; nucleotides 643, 1287, and 1290 are designated C, but may be C or G;nucleotides 772, 806, and 1261 are designated G, but may be A or G;nucleotides 1236, 1260, 1282, and 1289 are designated U, but may be G orT; residues 1247, 1257, 1293, and 1302 are designated C, but may be C orT; and nucleotides 1266 and 1298 are designated T, but may be A or T.Additional sequencing indicates that nucleotide 567 is A; 574 is G; 640is G; 742 is G; and 806 is G. Predicted signal cleavage is about betweenthr29 and asp30.TCGACCCACG CGTCCGCGCT GCGACTCAGA CCTCAGCTCC AACATATGCA TTCTGAAGAA   60AGATGGCTGA GATGGACAGA ATGCTTTATT TTGGAAAGAA ACAATGTTCT AGGTCAAACT  120GAGTCTACCA A ATG CAG ACT TTC ACA ATG GTT CTA GAA GAA ATC TGG ACA  170             Met Gln Thr Phe Thr Met Val Leu Glu Glu Ile Trp Thr               1               5                  10AGT CTT TTC ATG TGG TTT TTC TAC GCA TTG ATT CCA TGT TTG CTC ACA  218Ser Leu Phe Met Trp Phe Phe Tyr Ala Leu Ile Pro Cys Leu Leu Thr     15                  20                  25GAT GAA GTG GCC ATT CTG CCT GCC CCT CAG AAC CTC TCT GTA CTC TCA  266Asp Glu Val Ala Ile Leu Pro Ala Pro Gln Asn Leu Ser Val Leu Ser 30                  35                  40                  45ACC AAC ATG AAG CAT CTC TTG ATG TGG AGC CCA GTG ATC GCG CCT GGA  314Thr Asn Met Lys His Leu Leu Met Trp Ser Pro Val Ile Ala Pro Gly                 50                  55                  60GAA ACA GTG TAC TAT TCT GTC GAA TAC CAG GGG GAG TAC GAG AGC CTG  362Glu Thr Val Tyr Tyr Ser Val Glu Tyr Gln Gly Glu Tyr Glu Ser Leu             65                  70                  75TAC ACG AGC CAC ATC TGG ATC CCC AGC AGC TGG TGC TCA CTC ACT GAA  410Tyr Thr Ser His Ile Trp Ile Pro Ser Ser Trp Cys Ser Leu Thr Glu         80                  85                  90GGT CCT GAG TGT GAT GTC ACT GAT GAC ATC ACG GCC ACT GTG CCA TAC  458Gly Pro Glu Cys Asp Val Thr Asp Asp Ile Thr Ala Thr Val Pro Tyr     95                 100                 105AAC CTT CGT GTC AGG GCC ACA TTG GGC TCA CAG ACC TCA GCC TGG AGC  506Asn Leu Arg Val Arg Ala Thr Leu Gly Ser Gln Thr Ser Ala Trp Ser110                 115                 120                 125ATC CTG AAG CAT CCC TTT AAT AGA AAC TCA ACC ATC CTT ACC CGA CCT  554Ile Leu Lys His Pro Phe Asn Arg Asn Ser Thr Ile Leu Thr Arg Pro                130                 135                 140GGG ATG GAG ATC CCC AAA CAT GGC TTC CAC CTG GTT ATT GAG CTG GAG  602Gly Met Glu Ile Pro Lys His Gly Phe His Leu Val Ile Glu Leu Glu            145                 150                 155GAC CTG GGG CCC CAG TTT GAG TTC CTT GTG GCC TAC TGG ACG AGG GAG  650Asp Leu Gly Pro Gln Phe Glu Phe Leu Val Ala Tyr Trp Thr Arg Glu        160                 165                 170CCT GGT GCC GAG GAA CAT GTC AAA ATG GTG AGG AGT GGG GGT ATT CCA  698Pro Gly Ala Glu Glu His Val Lys Met Val Arg Ser Gly Gly Ile Pro    175                 180                 185GTG CAC CTA GAA ACC ATG GAG CCA GGG GCT GCA TAC TGT GTG AAG GCC  746Val His Leu Glu Thr Met Glu Pro Gly Ala Ala Tyr Cys Val Lys Ala190                 195                 200                 205CAG ACA TTC GTG AAG GCC ATT GGG AGG TAC AGC GCC TTC AGC CAG ACA  794Gln Thr Phe Val Lys Ala Ile Gly Arg Tyr Ser Ala Phe Ser Gln Thr                210                 215                 220GAA TGT GTG GAG GTG CAA GGA GAG GCC ATT CCC CTG GTA CTG GCC CTG  842Glu Cys Val Glu Val Gln Gly Glu Ala Ile Pro Leu Val Leu Ala Leu            225                 230                 235TTT GCC TTT GTT GGC TTC ATG CTG ATC CTT GTG GTC GTG CCA CTG TTC  890Phe Ala Phe Val Gly Phe Met Leu Ile Leu Val Val Val Pro Leu Phe        240                 245                 250GTC TGG AAA ATG GGC CGG CTG CTC CAG TAC TCC TGT TGC CCC GTG GTG  938Val Trp Lys Met Gly Arg Leu Leu Gln Tyr Ser Cys Cys Pro Val Val    255                 260                 265GTC CTC CCA GAC ACC TTG AAA ATA ACC AAT TCA CCC CAG AAG TTA ATC  986Val Leu Pro Asp Thr Leu Lys Ile Thr Asn Ser Pro Gln Lys Leu Ile270                 275                 280                 285AGC TGC AGA AGG GAG GAG GTG GAT GCC TGT GCC ACG GCT GTG ATG TCT 1034Ser Cys Arg Arg Glu Glu Val Asp Ala Cys Ala Thr Ala Val Met Ser                290                 295                 300CCT GAG GAA CTC CTC AGG GCC TGG ATC TCA TAGGTTTGCG GAAGGGCCCA 1084Pro Glu Glu Leu Leu Arg Ala Trp Ile Ser            305                 310GGTGAAGCCG AGAACCTGGT CTGCATGACA TGGAAACCAT GAGGGGACAA GTTGTGTTTC 1144TGTTTTCCGC CACGGACAAG GGATGAGAGA AGTAGGAAGA GCCTGTTGTC TACAAGTCTA 1204GAAGCAACCA TCAGAGGCAG GGTGGTTTGT CTAACAGAAC AACTGACTGA GGCTATGGGG 1264GTTGTGACCT CTAGACTTTG GGCTTCCACT TGCTTGGCTG AGCAACCCTG GGAAAAGTGA 1324CTTCATCCCT TCGGTCCCAA GTTTTCTCAT CTGTAATGGG GGATCCCTAC AAAACTG 1381

TABLE 2 Partial nucleotide and amino acid sequences of DNAX IFNReceptor Subunit like embodiments (DIRS2), originally designated HOFNY28(SEQ ID NO: 3 and 4). Nucleotide 193 designated C, may be C or T;additional sequencing indicates that nucleotide is C.C CGG GTC GAC CCA CGC GTC CGC CTG GTT TCC CCC TGG CTG ACA GTG   46  Arg Val Asp Pro Arg Val Arg Leu Val Ser Pro Trp Leu Thr Val    1               5                  10                  15CCT TGG TTC CTG TCC TGT TGG AAT GTT ACC ATT GGG CCT CCT GAG AGC   94Pro Trp Phe Leu Ser Cys Trp Asn Val Thr Ile Gly Pro Pro Glu Ser                 20                  25                  30ATC TGG GTG ACG CCG GGA GAA GCC TCC CTC ATC ATC AGG TTC TCC TCT  142Ile Trp Val Thr Pro Gly Glu Ala Ser Leu Ile Ile Arg Phe Ser Ser             35                  40                  45CCC TTC GAC GTC CCT CCC AAC CTG GGC TAT TTC CAG TAC TAT GTC CAT  190Pro Phe Asp Val Pro Pro Asn Leu Gly Tyr Phe Gln Tyr Tyr Val His         50                  55                  60TAC TGG GAA AAG GCG GGA ATC CAA AAG GTT AAA GGT CCT TTC AAG AGC  238Tyr Trp Glu Lys Ala Gly Ile Gln Lys Val Lys Gly Pro Phe Lys Ser     65                  70                  75AAC TCC ATC GTG TTG GAT GGC TTG AGA CCC TTA AGA GAA TAC TGT TTA  286Asn Ser Ile Val Leu Asp Gly Leu Arg Pro Leu Arg Glu Tyr Cys Leu 80                  85                  90                  95CAA GTG AAG GCG CAT CTC TTT CGC ACA TCC TGC AAC ACC TCT AGG CCC  334Gln Val Lys Ala His Leu Phe Arg Thr Ser Cys Asn Thr Ser Arg Pro                100                 105                 110GGC CGC TTA AGC AAC ATA ACT TGC TAC GAA ACA ATG ATG GAT GCC ACT  382Gly Arg Leu Ser Asn Ile Thr Cys Tyr Glu Thr Met Met Asp Ala Thr            115                 120                 125ACG AAG CTT CAA CAA GTC ATC CTC ATC GCC GTG GGA GTC TTT CTG TCG  430Thr Lys Leu Gln Gln Val Ile Leu Ile Ala Val Gly Val Phe Leu Ser        130                 135                 140CTG GCG GCG CTG GCG GGG GGC TGT TTC TTC CTG GTG CTG AGA TAC AAA  478Leu Ala Ala Leu Ala Gly Gly Cys Phe Phe Leu Val Leu Arg Tyr Lys    145                 150                 155GGC CTG GTG AAA TAC TGG TTT CAC TCT CCG CCA AGC ATC CCA TCA CAA  526Gly Leu Val Lys Tyr Trp Phe His Ser Pro Pro Ser Ile Pro Ser Gln160                 165                 170                 175ATC GAA GAG TAT CTG AAG GAC CCG AGC CAG CCT ATC CTA GAG GCC CTG  574Ile Glu Glu Tyr Leu Lys Asp Pro Ser Gln Pro Ile Leu Glu Ala Leu                180                 185                 190GAC AAG GAC ACG TCA CCA ACA GAT GAT GCC TGG GAC TTG GTG TCT GTT  622Asp Lys Asp Thr Ser Pro Thr Asp Asp Ala Trp Asp Leu Val Ser Val            195                 200                 205GTT GCA TTT CCA GCA AAG GAG CAA GAA GAT GTT CCC CAA AGC ACT TTG  670Val Ala Phe Pro Ala Lys Glu Gln Glu Asp Val Pro Gln Ser Thr Leu        210                 215                 220ACC CAA AAC TCT GGT GCG GTC TGC TAGCCTGTGG GGTAAGGGCT CTGAGCCGAG  724Thr Gln Asn Ser Gly Ala Val Cys     225                 230GAAGCTGCTG ATGTCCATGT CAGCACTTTA TGGAATCCGG TCCTCCATTT TCCTGTCCCC  784AAAAGGCCCG TCAGTGCCTG TGAAGATGTA ACGGGTCTCA TGGGGGCGAC AAGCTTATTG  844ATTTTTTTCT TCAAACTAAG AGTTTTCTAA TCATACGCGT TTTTAGAATA ATTCTACAGA  904TATGTCCCCG AAAGATTAAG ATTTCTCTTA AACACTAAAA AGACATGTAA TTATTTGTTA  964GCAAATGGGC GTCTGGCACG CCTCTGACAC TTTTTCGTCA GCAGCCAGGA CACGAGGTCC 1024CCTCCTTGAT GAAGCCCCTC GGGCAGACCA TGTCACCTGT CCCAGCCTGC CCCAAGAAGG 1084GACATTAAGT GGCCCTTCTT CATATCCAAA CACCTGGCTT GAAATGTGAT TAGCCCTGTA 1144AATAGTTTCA CAGAGATTAA GCCTTTTTTT CCCCCAAGTT AGGAATAAAA GACTATAATT 1204AACTTTTTAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1244

TABLE 3 Sequence alignment of related IFN receptor family members. DR1is a primate DIRS1 protein sequence; DR2 is a primate DIRS2 proteinsequence; the IRβ is the human IFN-γ receptor beta subunit (SEQ ID NO:5), see Soh, et al. (1994) Cell 76: 793-802; and CRF is the crf2-4 protein(SEQ ID NO: 6), see Lutfalla, et al. (1993) Genomics 16: 366-373: DR2---------- ---------- ---------- -------RVD PRVRLV---- ---------- DR1MQTFTMVLEE IWTSLFMWFF YALIPCLLTD EVAILPAPQN LSVLSTNMKH LLMWSPVIAP IRβ-----MRPTL LWSLLLLLGV FAAAAAAPPD PLSQLPAPQH PKIRLYNAEQ VLSWEPVALS crf---------M AWSLGSWLGG CLLVSALG-- ---MVPPPEN VRMNSVNFKN ILQWESPAFA DR2---------- ---------- ---------- ---------- ---------- --------SP DR1GETVYYSVEY QGEYES--LY TSHIWIPSSW CSLTEGPECD VTDDITAT-- ---VPYNLRV IRβNSTRPVVYRV QFKYTDSKWF TADIMSIGVN CTQITATECD FTAASPSAGF PMDFNVTLRL crfKGNLTFTAQY LSYR------ -----IFQDK CMNTTLTECD FSSLSKYG-- ----DHTLRV DR2WLTVPWFLSC WNVTIGPPES IWVTPGEASL IIRFSSPFDV PPN------- -LGYFQYYVH DR1RATLGSQTSA WSILK-HPFN RNSTILTRPG MEIXKXGFHL VIELE---DL GPQ------- IRβRAELGALHSA WVTMPWFQHY RNVTVGPPEN IEVTPGEGSL IIRFSSPFDI ADTS------ crfRAEFADEHSD WVNIT-FCPV DDTIIGPP-G MQVEVLADSL HMRFLAPKIE NEYETWTMKN DR2YW--EKAGIQ KVKGPFKSNS -IVLDGLRPL REYCLQVKAH LFRTSCNTSR PGRLSNITCY DR1----FEFLVA YWXREPGAEE HVKMVRSGGI PVHLETMEPG AAYCVKAQT- -FVKAIGX-- IRβ-TAFFCYYVH Y--WEKGGIQ QVKGPFRSNS -ISLDNLKPS RVYCLQVQAQ LLWNKSNIFR crfVYNSWTYNVQ YW--KNGTDE KFQITPQYDF -EVLRNLEPW TTYCVQVRG- -FLPDRNK-- DR2ETMMDATTKL QQVILIAVGV FLSLAALAGG CFFLVLRYKG LVKYWFHSPP SIPSQIEEYL DR1YSAFSQTECV EVQG-EAIPL VLALFAFVG- -FMLILVVVP LF--VWKMGR LLQYSCCPVV IRβVGHLSNISCY ETMADASTEL QQVILISVGT FSLLSVLAGA CFFLVLKYRG LIKYWFHTPP crfAGEWSEPVCE QTTHDETVPS WMVAVILMAS VFMVCLALLG CFSLLWCVYK KTKYAFSPRN DR2KDPSQPILEA LDKDTSPTDD AWDLVSVVAF PAK--EQE-- DVPQSTLTQN DR1VLPDTLKITN S-P-QKLISC R----REEVD AC--ATAVMS PEE------- IRβSIPLQIEEYL KDPTQPILEA LDKDSSPKDD VWDSVSIISF PEK--EQE-- crfSLPQHLKEFL GHPHHNTLLF FSFPLSDEND VFDKLSVIAE DSESGKQNPG DR2 SGAVC DR1-LLRAWIS IRβ DVLQTL crf DSCSLGTPPG QGPQS

Table 3 shows comparison of the available sequences of primateembodiments of DIRS1, DIRS2, and two related interferon receptor familymembers. Both of the new DIRS appear to exhibit sequence similarity tobeta interferon receptor subunits.

Structural features of the human DIRS1, and similarly for the otherreceptors as aligned in Table 3, include characteristic transmembranesegments of the IRβ and crf from 261-273, and correspond to: from aboutval1 to pro133; fibronectin domains corresponding to the DIRS1 sequencefrom about gly134 to pro232, gly233 to gly306, and pro307 to lys403; atransmembrane segment from about val404 to gly427; and an intracellulardomain from about arg428 to the carboxy terminus. Of particular interestis the WGEWS motif corresponding to residues trp104 to ser108.

As used herein, the term DIRS1 shall be used to describe a proteincomprising a protein or peptide segment having or sharing the amino acidsequence shown in Table 1, or a substantial fragment thereof. Theinvention also includes a protein variation of the respective DIRS1allele whose sequence is provided, e.g., a mutein or solubleextracellular construct. Typically, such agonists or antagonists willexhibit less than about 10% sequence differences, and thus will oftenhave between 1- and 11-fold substitutions, e.g., 2-, 3-, 5-, 7-fold, andothers. It also encompasses allelic and other variants, e.g., naturalpolymorphic, of the protein described. Typically, it will bind to itscorresponding biological ligand, perhaps in a dimerized state with analpha receptor subunit, with high affinity, e.g., at least about 100 nM,usually better than about 30 nM, preferably better than about 10 nM, andmore preferably at better than about 3 nM. The term shall also be usedherein to refer to related naturally occurring forms, e.g., alleles,polymorphic variants, and metabolic variants of the mammalian protein.

This invention also encompasses proteins or peptides having substantialamino acid sequence identity with the amino acid sequence in Table 1. Itwill include sequence variants with relatively few substitutions, e.g.,preferably less than about 3-5. Other embodiments include forms inassociation with an alpha subunit, e.g., a DSRS1, and/or with ligand,e.g., DIL-30.

A substantial polypeptide “fragment”, or “segment”, is a stretch ofamino acid residues of at least about 8 amino acids, generally at least10 amino acids, more generally at least 12 amino acids, often at least14 amino acids, more often at least 16 amino acids, typically at least18 amino acids, more typically at least 20 amino acids, usually at least22 amino acids, more usually at least 24 amino acids, preferably atleast 26 amino acids, more preferably at least 28 amino acids, and, inparticularly preferred embodiments, at least about 30 or more aminoacids, e.g., 35, 40, 50, 70, 90, 110, etc. Specific ends may be at allpossible or appropriate combinations, or at proline residues. Sequencesof segments of different proteins can be compared to one another overappropriate length stretches.

The invention provides polypeptides exhibiting a plurality of distinct,e.g., nonoverlapping, segments of the specified length. Typically, theplurality will be at least two, more usually at least three, andpreferably 5, 7, or even more. While the length minima are provided,longer lengths, of various sizes, may be appropriate, e.g., one oflength 7, and two of length 12.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches. In some comparisons, gaps may be introduces,as required. See, e.g., Needleham, et al. (1970) J. Mol. Biol.48:443-453; Sankoff, et al., (1983) chapter one in Time Warps, StringEdits, and Macromolecules: The Theory and Practice of SequenceComparison, Addison-Wesley, Reading, Mass.; and software packages fromNCBI, NIH; and the University of Wisconsin Genetics Computer Group(GCG), Madison, Wis.; each of which is incorporated herein by reference.This changes when considering conservative substitutions as matches.Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. Homologous amino acidsequences are intended to include natural allelic and interspeciesvariations in the cytokine sequence. Typical homologous proteins orpeptides will have from 50-100% homology (if gaps can be introduced), to60-100% homology (if conservative substitutions are included) with anamino acid sequence segment of Table 1. Homology measures will be atleast about 70%, generally at least 76%, more generally at least 81%,often at least 85%, more often at least 88%, typically at least 90%,more typically at least 92%, usually at least 94%, more usually at least95%, preferably at least 96%, and more preferably at least 97%, and inparticularly preferred embodiments, at least 98% or more. The degree ofhomology will vary with the length of the compared segments. Homologousproteins or peptides, such as the allelic variants, will share mostbiological activities with the embodiments described in Table 1.

As used herein, the term “biological activity” is used to describe,without limitation, effects on inflammatory responses, innate immunity,and/or morphogenic development by cytokine-like ligands. For example,these receptors should mediate phosphatase or phosphorylase activities,which activities are easily measured by standard procedures. See, e.g.,Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II,Academic Press, San Diego, Calif.; Hanks, et al. (1991) Meth. Enzymol.200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell61:743-752; Pines, et al. (1991) Cold Spring Harbor Symp. Quant. Biol.56:449-463; and Parker, et al. (1993) Nature 363:736-738. The receptors,or portions thereof, may be useful as phosphate labeling enzymes tolabel general or specific substrates.

The terms ligand, agonist, antagonist, and analog of, e.g., a DIRS1,include molecules that modulate the characteristic cellular responses tocytokine ligand proteins, as well as molecules possessing the morestandard structural binding competition features of ligand-receptorinteractions, e.g., where the receptor is a natural receptor or anantibody. The cellular responses likely are typically mediated throughreceptor tyrosine kinase pathways.

Also, a ligand is a molecule which serves either as a natural ligand towhich said receptor, or an analog thereof, binds, or a molecule which isa functional analog of the natural ligand. The functional analog may bea ligand with structural modifications, or may be a wholly unrelatedmolecule which has a molecular shape which interacts with theappropriate ligand binding determinants. The ligands may serve asagonists or antagonists, see, e.g., Goodman, et al. (eds. 1990) Goodman& Gilman's: The Pharmacological Bases of Therapeutics, Pergamon Press,New York.

Rational drug design may also be based upon structural studies of themolecular shapes of a receptor or antibody and other effectors orligands. See, e.g., Herz, et al. (1997) J. Recept. Signal Transduct.Res. 17:671-776; and Chaiken, et al. (1996) Trends Biotechnol.14:369-375. Effectors may be other proteins which mediate otherfunctions in response to ligand binding, or other proteins whichnormally interact with the receptor. One means for determining whichsites interact with specific other proteins is a physical structuredetermination, e.g., x-ray crystallography or 2 dimensional NMRtechniques. These will provide guidance as to which amino acid residuesform molecular contact regions. For a detailed description of proteinstructural determination, see, e.g., Blundell and Johnson (1976) ProteinCrystallography, Academic Press, New York, which is hereby incorporatedherein by reference.

II. Activities

The cytokine receptor-like proteins will have a number of differentbiological activities, e.g., modulating cell proliferation, or inphosphate metabolism, being added to or removed from specificsubstrates, typically proteins. Such will generally result in modulationof an inflammatory function, other innate immunity response, or amorphological effect. The subunit will probably have a specific lowaffinity binding to the ligand.

The DIRS1 has the characteristic motifs of a receptor signaling throughthe JAK pathway. See, e.g., Ihle, et al. (1997) Stem Cells 15 (suppl.1):105-111; Silvennoinen, et al. (1997) APMIS 105:497-509; Levy (1997)Cytokine Growth Factor Review 8:81-90; Winston and Hunter (1996) CurrentBiol. 6:668-671; Barrett (1996) Baillieres Clin. Gastroenterol. 10:1-15;and Briscoe, et al. (1996) Philos. Trans. R. Soc. Lond. B. Biol. Sci.351:167-171.

The biological activities of the cytokine receptor subunits will berelated to addition or removal of phosphate moieties to substrates,typically in a specific manner, but occasionally in a non specificmanner. Substrates may be identified, or conditions for enzymaticactivity may be assayed by standard methods, e.g., as described inHardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II,Academic Press, San Diego, Calif.; Hanks, et al. (1991) Meth. Enzymol.200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell61:743-752; Pines, et al. (1991) Cold Spring Harbor Symp. Quant. Biol.56:449-463; and Parker, et al. (1993) Nature 363:736-738.

III. Nucleic Acids

This invention contemplates use of isolated nucleic acid or fragments,e.g., which encode these or closely related proteins, or fragmentsthereof, e.g., to encode a corresponding polypeptide, preferably onewhich is biologically active. In addition, this invention coversisolated or recombinant DNAs which encode such proteins or polypeptideshaving characteristic sequences of the DIRS1s. Typically, the nucleicacid is capable of hybridizing, under appropriate conditions, with anucleic acid sequence segment shown in Table 1, but preferably not witha corresponding segment of other receptors described in Table 3. Saidbiologically active protein or polypeptide can be a full length protein,or fragment, and will typically have a segment of amino acid sequencehighly homologous, e.g., exhibiting significant stretches of identity,to one shown in Table 1. Further, this invention covers the use ofisolated or recombinant nucleic acid, or fragments thereof, which encodeproteins having fragments which are equivalent to the DIRS1 proteins.The isolated nucleic acids can have the respective regulatory sequencesin the 5′ and 3′ flanks, e.g., promoters, enhancers, poly-A additionsignals, and others from the natural gene.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially pure, e.g., separated from othercomponents which naturally accompany a native sequence, such asribosomes, polymerases, and flanking genomic sequences from theoriginating species. The term embraces a nucleic acid sequence which hasbeen removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates, which are thereby distinguishablefrom naturally occurring compositions, and chemically synthesizedanalogs or analogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule,either completely or substantially pure.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain heterogeneity,preferably minor. This heterogeneity is typically found at the polymerends or portions not critical to a desired biological function oractivity.

A “recombinant” nucleic acid is typically defined either by its methodof production or its structure. In reference to its method ofproduction, e.g., a product made by a process, the process is use ofrecombinant nucleic acid techniques, e.g., involving human interventionin the nucleotide sequence. Typically this intervention involves invitro manipulation, although under certain circumstances it may involvemore classical animal breeding techniques. Alternatively, it can be anucleic acid made by generating a sequence comprising fusion of twofragments which are not naturally contiguous to each other, but is meantto exclude products of nature, e.g., naturally occurring mutants asfound in their natural state. Thus, for example, products made bytransforming cells with an unnaturally occurring vector is encompassed,as are nucleic acids comprising sequence derived using any syntheticoligonucleotide process. Such a process is often done to replace a codonwith a redundant codon encoding the same or a conservative amino acid,while typically introducing or removing a restriction enzyme sequencerecognition site. Alternatively, the process is performed to jointogether nucleic acid segments of desired functions to generate a singlegenetic entity comprising a desired combination of functions not foundin the commonly available natural forms, e.g., encoding a fusionprotein. Restriction enzyme recognition sites are often the target ofsuch artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. This will include a dimeric repeat. Specifically includedare synthetic nucleic acids which, by genetic code redundancy, encodeequivalent polypeptides to fragments of DIRS1 and fusions of sequencesfrom various different related molecules, e.g., other cytokine receptorfamily members.

A “fragment” in a nucleic acid context is a contiguous segment of atleast about 17 nucleotides, generally at least 21 nucleotides, moregenerally at least 25 nucleotides, ordinarily at least 30 nucleotides,more ordinarily at least 35 nucleotides, often at least 39 nucleotides,more often at least 45 nucleotides, typically at least 50 nucleotides,more typically at least 55 nucleotides, usually at least 60 nucleotides,more usually at least 66 nucleotides, preferably at least 72nucleotides, more preferably at least 79 nucleotides, and inparticularly preferred embodiments will be at least 85 or morenucleotides. Typically, fragments of different genetic sequences can becompared to one another over appropriate length stretches, particularlydefined segments such as the domains described below.

A nucleic acid which codes for a DIRS1 will be particularly useful toidentify genes, mRNA, and cDNA species which code for itself or closelyrelated proteins, as well as DNAs which code for polymorphic, allelic,or other genetic variants, e.g., from different individuals or relatedspecies. Preferred probes for such screens are those regions of theinterleukin which are conserved between different polymorphic variantsor which contain nucleotides which lack specificity, and will preferablybe full length or nearly so. In other situations, polymorphic variantspecific sequences will be more useful.

This invention further covers recombinant nucleic acid molecules andfragments having a nucleic acid sequence identical to or highlyhomologous to the isolated DNA set forth herein. In particular, thesequences will often be operably linked to DNA segments which controltranscription, translation, and DNA replication. These additionalsegments typically assist in expression of the desired nucleic acidsegment.

Homologous, or highly identical, nucleic acid sequences, when comparedto one another, e.g., DIRS1 sequences, exhibit significant similarity.The standards for homology in nucleic acids are either measures forhomology generally used in the art by sequence comparison or based uponhybridization conditions. Comparative hybridization conditions aredescribed in greater detail below.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optical alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2:482, by the homology alignment algorithm of Needlman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendrogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins and Sharp (1989) CABIOS 5:151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul, et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction are halted when: the cumulative alignment score fallsoff by the quantity X from its maximum achieved value; the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences of polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.Hybridization under stringent conditions should give a background of atleast 2-fold over background, preferably at least 3-5 or more.

Substantial identity in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 60% of thenucleotides, generally at least 66%, ordinarily at least 71%, often atleast 76%, more often at least 80%, usually at least 84%, more usuallyat least 88%, typically at least 91%, more typically at least about 93%,preferably at least about 95%, more preferably at least about 96 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides, including, e.g., segments encoding structural domainssuch as the segments described below. Alternatively, substantialidentity will exist when the segments will hybridize under selectivehybridization conditions, to a strand or its complement, typically usinga sequence derived from Table 1. Typically, selective hybridization willoccur when there is at least about 55% homology over a stretch of atleast about 14 nucleotides, more typically at least about 65%,preferably at least about 75%, and more preferably at least about 90%.See, Kanehisa (1984) Nucl. Acids Res. 12:203-213, which is incorporatedherein by reference. The length of homology comparison, as described,may be over longer stretches, and in certain embodiments will be over astretch of at least about 17 nucleotides, generally at least about 20nucleotides, ordinarily at least about 24 nucleotides, usually at leastabout 28 nucleotides, typically at least about 32 nucleotides, moretypically at least about 40 nucleotides, preferably at least about 50nucleotides, and more preferably at least about 75 to 100 or morenucleotides. This includes, e.g., 125, 150, 175, 200, 225, 246, 273, andother lengths.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters typically controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., more usually in excessof about 37° C., typically in excess of about 45° C., more typically inexcess of about 55° C., preferably in excess of about 65° C., and morepreferably in excess of about 70° C. Stringent salt conditions willordinarily be less than about 500 mM, usually less than about 400 mM,more usually less than about 300 mM, typically less than about 200 mM,preferably less than about 100 mM, and more preferably less than about80 mM, even down to less than about 20 mM. However, the combination ofparameters is much more important than the measure of any singleparameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370, which is hereby incorporated herein by reference.

The isolated DNA can be readily modified by nucleotide substitutions,nucleotide deletions, nucleotide insertions, and inversions ofnucleotide stretches. These modifications result in novel DNA sequenceswhich encode this protein or its derivatives. These modified sequencescan be used to produce mutant proteins (muteins) or to enhance theexpression of variant species. Enhanced expression may involve geneamplification, increased transcription, increased translation, and othermechanisms. Such mutant DIRS1-like derivatives include predetermined orsite-specific mutations of the protein or its fragments, includingsilent mutations using genetic code degeneracy. “Mutant DIRS1” as usedherein encompasses a polypeptide otherwise falling within the homologydefinition of the DIRS1 as set forth above, but having an amino acidsequence which differs from that of other cytokine receptor-likeproteins as found in nature, whether by way of deletion, substitution,or insertion. In particular, “site specific mutant DIRS1” encompasses aprotein having substantial sequence identity with a protein of Table 1,and typically shares most of the biological activities or effects of theforms disclosed herein.

Although site specific mutation sites are predetermined, mutants neednot be site specific. Mammalian DIRS1 mutagenesis can be achieved bymaking amino acid insertions or deletions in the gene, coupled withexpression. Substitutions, deletions, insertions, or many combinationsmay be generated to arrive at a final construct. Insertions includeamino- or carboxy-terminal fusions. Random mutagenesis can be conductedat a target codon and the expressed mammalian DIRS1 mutants can then bescreened for the desired activity, providing some aspect of astructure-activity relationship. Methods for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown in the art, e.g., by M13 primer mutagenesis. See also Sambrook, etal. (1989) and Ausubel, et al. (1987 and periodic Supplements).

The mutations in the DNA normally should not place coding sequences outof reading frames and preferably will not create complementary regionsthat could hybridize to produce secondary mRNA structure such as loopsor hairpins.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

Polymerase chain reaction (PCR) techniques can often be applied inmutagenesis. Alternatively, mutagenesis primers are commonly usedmethods for generating defined mutations at predetermined sites. See,e.g., Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif.; and Dieffenbach andDveksler (eds. 1995) PCR Primer: A Laboratory Manual Cold Spring HarborPress, CSH, NY.

IV. Proteins, Peptides

As described above, the present invention encompasses primate DIRS1,e.g., whose sequences are disclosed in Table 1, and described above.Allelic and other variants are also contemplated, including, e.g.,fusion proteins combining portions of such sequences with others,including epitope tags and functional domains.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these rodent proteins.A heterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. Thus, the fusionproduct of a DIRS1 with another cytokine receptor is a continuousprotein molecule having sequences fused in a typical peptide linkage,typically made as a single translation product and exhibitingproperties, e.g., sequence or antigenicity, derived from each sourcepeptide. A similar concept applies to heterologous nucleic acidsequences.

In addition, new constructs may be made from combining similarfunctional or structural domains from other related proteins, e.g.,cytokine receptors or Toll-like receptors, including species variants.For example, ligand-binding or other segments may be “swapped” betweendifferent new fusion polypeptides or fragments. See, e.g., Cunningham,et al. (1989) Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol.Chem. 263:15985-15992, each of which is incorporated herein byreference. Thus, new chimeric polypeptides exhibiting new combinationsof specificities will result from the functional linkage ofreceptor-binding specificities. For example, the ligand binding domainsfrom other related receptor molecules may be added or substituted forother domains of this or related proteins. The resulting protein willoften have hybrid function and properties. For example, a fusion proteinmay include a targeting domain which may serve to provide sequesteringof the fusion protein to a particular subcellular organelle.

Candidate fusion partners and sequences can be selected from varioussequence data bases, e.g., GenBank; NCBI, NIH; and BCG, University ofWisconsin Biotechnology Computing Group, Madison, Wis., which are eachincorporated herein by reference.

The present invention particularly provides muteins which bindcytokine-like ligands, and/or which are affected in signal transduction.Structural alignment of human DIRS1 with other members of the cytokinereceptor family show conserved features/residues. See Table 3. Alignmentof the human DIRS1 sequence with other members of the cytokine receptorfamily indicates various structural and functionally shared features.See also, Bazan, et al. (1996) Nature 379:591; Lodi, et al. (1994)Science 263:1762-1766; Sayle and Milner-White (1995) TIBS 20:374-376;and Gronenberg, et al. (1991) Protein Engineering 4:263-269.

Substitutions with either mouse sequences or human sequences areparticularly preferred. Conversely, conservative substitutions away fromthe ligand binding interaction regions will probably preserve mostsignaling activities; and conservative substitutions away from theintracellular domains will probably preserve most ligand bindingproperties.

“Derivatives” of the primate DIRS1 include amino acid sequence mutants,glycosylation variants, metabolic derivatives and covalent oraggregative conjugates with other chemical moieties. Covalentderivatives can be prepared by linkage of functionalities to groupswhich are found in the DIRS1 amino acid side chains or at the N- orC-termini, e.g., by means which are well known in the art. Thesederivatives can include, without limitation, aliphatic esters or amidesof the carboxyl terminus, or of residues containing carboxyl sidechains, O-acyl derivatives of hydroxyl group-containing residues, andN-acyl derivatives of the amino terminal amino acid or amino-groupcontaining residues, e.g., lysine or arginine. Acyl groups are selectedfrom the group of alkyl-moieties, including C3 to C18 normal alkyl,thereby forming alkanoyl aroyl species.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

A major group of derivatives are covalent conjugates of the receptors orfragments thereof with other proteins of polypeptides. These derivativescan be synthesized in recombinant culture such as N- or C-terminalfusions or by the use of agents known in the art for their usefulness incross-linking proteins through reactive side groups. Preferredderivatization sites with cross-linking agents are at free amino groups,carbohydrate moieties, and cysteine residues.

Fusion polypeptides between the receptors and other homologous orheterologous proteins are also provided. Homologous polypeptides may befusions between different receptors, resulting in, for instance, ahybrid protein exhibiting binding specificity for multiple differentcytokine ligands, or a receptor which may have broadened or weakenedspecificity of substrate effect. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. Typical examples are fusions of areporter polypeptide, e.g., luciferase, with a segment or domain of areceptor, e.g., a ligand-binding segment, so that the presence orlocation of a desired ligand may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609, which is hereby incorporated herein byreference. Other gene fusion partners include glutathione-S-transferase(GST), bacterial β-galactosidase, trpE, Protein A, β-lactamase, alphaamylase, alcohol dehydrogenase, and yeast alpha mating factor. See,e.g., Godowski, et al. (1988) Science 241:812-816.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

Such polypeptides may also have amino acid residues which have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties, particularly those which havemolecular shapes similar to phosphate groups. In some embodiments, themodifications will be useful labeling reagents, or serve as purificationtargets, e.g., affinity ligands.

Fusion proteins will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, for example,in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2ded.), Vols. 1-3, Cold Spring Harbor Laboratory, and Ausubel, et al.(eds. 1987 and periodic supplements) Current Protocols in MolecularBiology, Greene/Wiley, New York, which are each incorporated herein byreference. Techniques for synthesis of polypeptides are described, forexample, in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156;Merrifield (1986) Science 232: 341-347; and Atherton, et al. (1989)Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford;each of which is incorporated herein by reference. See also Dawson, etal. (1994) Science 266:776-779 for methods to make larger polypeptides.

This invention also contemplates the use of derivatives of a DIRS1 otherthan variations in amino acid sequence or glycosylation. Suchderivatives may involve covalent or aggregative association withchemical moieties. These derivatives generally fall into three classes:(1) salts, (2) side chain and terminal residue covalent modifications,and (3) adsorption complexes, for example with cell membranes. Suchcovalent or aggregative derivatives are useful as immunogens, asreagents in immunoassays, or in purification methods such as foraffinity purification of a receptor or other binding molecule, e.g., anantibody. For example, a cytokine ligand can be immobilized by covalentbonding to a solid support such as cyanogen bromide-activated Sepharose,by methods which are well known in the art, or adsorbed onto polyolefinsurfaces, with or without glutaraldehyde cross-linking, for use in theassay or purification of an cytokine receptor, antibodies, or othersimilar molecules. The ligand can also be labeled with a detectablegroup, for example radioiodinated by the chloramine T procedure,covalently bound to rare earth chelates, or conjugated to anotherfluorescent moiety for use in diagnostic assays.

An DIRS1 of this invention can be used as an immunogen for theproduction of antisera or antibodies specific, e.g., capable ofdistinguishing between other cytokine receptor family members, for theDIRS1 or various fragments thereof. The purified DIRS1 can be used toscreen monoclonal antibodies or antigen-binding fragments prepared byimmunization with various forms of impure preparations containing theprotein. Antibodies can typically be substituted with antigen bindingfragments of natural antibodies, e.g., Fab, Fab2, Fv, etc. The purifiedDIRS1 can also be used as a reagent to detect antibodies generated inresponse to the presence of elevated levels of expression, orimmunological disorders which lead to antibody production to theendogenous receptor. Additionally, DIRS1 fragments may also serve asimmunogens to produce the antibodies of the present invention, asdescribed immediately below. For example, this invention contemplatesantibodies having binding affinity to or being raised against the aminoacid sequences shown in Table 1, fragments thereof, or varioushomologous peptides. In particular, this invention contemplatesantibodies having binding affinity to, or having been raised against,specific fragments which are predicted to be, or actually are, exposedat the exterior protein surface of the native DIRS1.

The blocking of physiological response to the receptor ligands mayresult from the inhibition of binding of the ligand to the receptor,likely through competitive inhibition. Thus, in vitro assays of thepresent invention will often use antibodies or antigen binding segmentsof these antibodies, or fragments attached to solid phase substrates.These assays will also allow for the diagnostic determination of theeffects of either ligand binding region mutations and modifications, orother mutations and modifications, e.g., which affect signaling orenzymatic function.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to the receptor or fragmentscompete with a test compound for binding to a ligand or other antibody.In this manner, the neutralizing antibodies or fragments can be used todetect the presence of a polypeptide which shares one or more bindingsites to a receptor and can also be used to occupy binding sites on areceptor that might otherwise bind a ligand.

V. Making Nucleic Acids and Protein

DNA which encodes the protein or fragments thereof can be obtained bychemical synthesis, screening cDNA libraries, or by screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.Natural sequences can be isolated using standard methods and thesequences provided herein, e.g., in Table 1. Other species counterpartscan be identified by hybridization techniques, or by various PCRtechniques, combined with or by searching in sequence databases, e.g.,GenBank.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length receptor or fragments which can in turn, forexample, be used to generate polyclonal or monoclonal antibodies; forbinding studies; for construction and expression of modified ligandbinding or kinase/phosphatase domains; and for structure/functionstudies. Variants or fragments can be expressed in host cells that aretransformed or transfected with appropriate expression vectors. Thesemolecules can be substantially free of protein or cellular contaminants,other than those derived from the recombinant host, and therefore areparticularly useful in pharmaceutical compositions when combined with apharmaceutically acceptable carrier and/or diluent. The protein, orportions thereof, may be expressed as fusions with other proteins.

Expression vectors are typically self-replicating DNA or RNA constructscontaining the desired receptor gene or its fragments, usually operablylinked to suitable genetic control elements that are recognized in asuitable host cell. These control elements are capable of effectingexpression within a suitable host. The specific type of control elementsnecessary to effect expression will depend upon the eventual host cellused. Generally, the genetic control elements can include a prokaryoticpromoter system or a eukaryotic promoter expression control system, andtypically include a transcriptional promoter, an optional operator tocontrol the onset of transcription, transcription enhancers to elevatethe level of mRNA expression, a sequence that encodes a suitableribosome binding site, and sequences that terminate transcription andtranslation. Expression vectors also usually contain an origin ofreplication that allows the vector to replicate independently of thehost cell.

The vectors of this invention include those which contain DNA whichencodes a protein, as described, or a fragment thereof encoding abiologically active equivalent polypeptide. The DNA can be under thecontrol of a viral promoter and can encode a selection marker. Thisinvention further contemplates use of such expression vectors which arecapable of expressing eukaryotic cDNA coding for such a protein in aprokaryotic or eukaryotic host, where the vector is compatible with thehost and where the eukaryotic cDNA coding for the receptor is insertedinto the vector such that growth of the host containing the vectorexpresses the cDNA in question. Usually, expression vectors are designedfor stable replication in their host cells or for amplification togreatly increase the total number of copies of the desirable gene percell. It is not always necessary to require that an expression vectorreplicate in a host cell, e.g., it is possible to effect transientexpression of the protein or its fragments in various hosts usingvectors that do not contain a replication origin that is recognized bythe host cell. It is also possible to use vectors that cause integrationof the protein encoding portion or its fragments into the host DNA byrecombination.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. Expressionvectors are specialized vectors which contain genetic control elementsthat effect expression of operably linked genes. Plasmids are the mostcommonly used form of vector but all other forms of vectors which servean equivalent function and which are, or become, known in the art aresuitable for use herein. See, e.g., Pouwels, et al. (1985 andSupplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., andRodriguez, et al. (eds. 1988) Vectors: A Survey of Molecular CloningVectors and Their Uses, Buttersworth, Boston, which are incorporatedherein by reference.

Transformed cells are cells, preferably mammalian, that have beentransformed or transfected with receptor vectors constructed usingrecombinant DNA techniques. Transformed host cells usually express thedesired protein or its fragments, but for purposes of cloning,amplifying, and manipulating its DNA, do not need to express the subjectprotein. This invention further contemplates culturing transformed cellsin a nutrient medium, thus permitting the receptor to accumulate in thecell membrane. The protein can be recovered, either from the culture or,in certain instances, from the culture medium.

For purposes of this invention, nucleic sequences are operably linkedwhen they are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression.

Suitable host cells include prokaryotes, lower eukaryotes, and highereukaryotes. Prokaryotes include both gram negative and gram positiveorganisms, e.g., E. coli and B. subtilis. Lower eukaryotes includeyeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express thereceptor or its fragments include, but are not limited to, such vectorsas those containing the lac promoter (pUC-series); trp promoter(pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pR promoters(pOTS); or hybrid promoters such as ptac (pDR540). See Brosius, et al.(1988) “Expression Vectors Employing Lambda-, trp-, lac-, andIpp-derived Promoters”, in Vectors: A Survey of Molecular CloningVectors and Their Uses, (eds. Rodriguez and Denhardt), Buttersworth,Boston, Chapter 10, pp. 205-236, which is incorporated herein byreference.

Lower eukaryotes, e.g., yeasts and Dictyostelium, may be transformedwith DIRS1 sequence containing vectors. For purposes of this invention,the most common lower eukaryotic host is the baker's yeast,Saccharomyces cerevisiae. It will be used to generically represent lowereukaryotes although a number of other strains and species are alsoavailable. Yeast vectors typically consist of a replication origin(unless of the integrating type), a selection gene, a promoter, DNAencoding the receptor or its fragments, and sequences for translationtermination, polyadenylation, and transcription termination. Suitableexpression vectors for yeast include such constitutive promoters as3-phosphoglycerate kinase and various other glycolytic enzyme genepromoters or such inducible promoters as the alcohol dehydrogenase 2promoter or metallothionine promoter. Suitable vectors includederivatives of the following types: self-replicating low copy number(such as the YRp-series), self-replicating high copy number (such as theYEp-series); integrating types (such as the YIp-series), ormini-chromosomes (such as the YCp-series).

Higher eukaryotic tissue culture cells are normally the preferred hostcells for expression of the functionally active interleukin protein. Inprinciple, many higher eukaryotic tissue culture cell lines areworkable, e.g., insect baculovirus expression systems, whether from aninvertebrate or vertebrate source. However, mammalian cells arepreferred. Transformation or transfection and propagation of such cellshas become a routine procedure. Examples of useful cell lines includeHeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney(BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS)cell lines. Expression vectors for such cell lines usually include anorigin of replication, a promoter, a translation initiation site, RNAsplice sites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as from adenovirus, SV40, parvoviruses, vacciniavirus, or cytomegalovirus. Representative examples of suitableexpression vectors include pcDNA1; pCD, see Okayama, et al. (1985) Mol.Cell Biol. 5:1136-1142; pMC1neo PolyA, see Thomas, et al. (1987) Cell51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.

For secreted proteins, an open reading frame usually encodes apolypeptide that consists of a mature or secreted product covalentlylinked at its N-terminus to a signal peptide. The signal peptide iscleaved prior to secretion of the mature, or active, polypeptide. Thecleavage site can be predicted with a high degree of accuracy fromempirical rules, e.g., von-Heijne (1986) Nucleic Acids Research14:4683-4690 and Nielsen, et al. (1997) Protein Eng. 10:1-12, and theprecise amino acid composition of the signal peptide often does notappear to be critical to its function, e.g., Randall, et al. (1989)Science 243:1156-1159; Kaiser et al. (1987) Science 235:312-317.

It will often be desired to express these polypeptides in a system whichprovides a specific or defined glycosylation pattern. In this case, theusual pattern will be that provided naturally by the expression system.However, the pattern will be modifiable by exposing the polypeptide,e.g., an unglycosylated form, to appropriate glycosylating proteinsintroduced into a heterologous expression system. For example, thereceptor gene may be co-transformed with one or more genes encodingmammalian or other glycosylating enzymes. Using this approach, certainmammalian glycosylation patterns will be achievable in prokaryote orother cells.

The source of DIRS1 can be a eukaryotic or prokaryotic host expressingrecombinant DIRS1, such as is described above. The source can also be acell line such as mouse Swiss 3T3 fibroblasts, but other mammalian celllines are also contemplated by this invention, with the preferred cellline being from the human species.

Now that the sequences are known, the primate DIRS1, fragments, orderivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young (1984) Solid Phase Peptide Synthesis, Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice ofPeptide Synthesis, Springer-Verlag, New York; and Bodanszky (1984) ThePrinciples of Peptide Synthesis, Springer-Verlag, New York; all of eachwhich are incorporated herein by reference. For example, an azideprocess, an acid chloride process, an acid anhydride process, a mixedanhydride process, an active ester process (for example, p-nitrophenylester, N-hydroxysuccinimide ester, or cyanomethyl ester), acarbodiimidazole process, an oxidative-reductive process, or adicyclohexylcarbodiimide (DCCD)/additive process can be used. Solidphase and solution phase syntheses are both applicable to the foregoingprocesses. Similar techniques can be used with partial DIRS1 sequences.

The DIRS1 proteins, fragments, or derivatives are suitably prepared inaccordance with the above processes as typically employed in peptidesynthesis, generally either by a so-called stepwise process whichcomprises condensing an amino acid to the terminal amino acid, one byone in sequence, or by coupling peptide fragments to the terminal aminoacid. Amino groups that are not being used in the coupling reactiontypically must be protected to prevent coupling at an incorrectlocation.

If a solid phase synthesis is adopted, the C-terminal amino acid isbound to an insoluble carrier or support through its carboxyl group. Theinsoluble carrier is not particularly limited as long as it has abinding capability to a reactive carboxyl group. Examples of suchinsoluble carriers include halomethyl resins, such as chloromethyl resinor bromomethyl resin, hydroxymethyl resins, phenol resins,tert-alkyloxycarbonylhydrazidated resins, and the like.

An amino group-protected amino acid is bound in sequence throughcondensation of its activated carboxyl group and the reactive aminogroup of the previously formed peptide or chain, to synthesize thepeptide step by step. After synthesizing the complete sequence, thepeptide is split off from the insoluble carrier to produce the peptide.This solid-phase approach is generally described by Merrifield, et al.(1963) in J. Am. Chem. Soc. 85:2149-2156, which is incorporated hereinby reference.

The prepared protein and fragments thereof can be isolated and purifiedfrom the reaction mixture by means of peptide separation, for example,by extraction, precipitation, electrophoresis, various forms ofchromatography, and the like. The receptors of this invention can beobtained in varying degrees of purity depending upon desired uses.Purification can be accomplished by use of the protein purificationtechniques disclosed herein, see below, or by the use of the antibodiesherein described in methods of immunoabsorbant affinity chromatography.This immunoabsorbant affinity chromatography is carried out by firstlinking the antibodies to a solid support and then contacting the linkedantibodies with solubilized lysates of appropriate cells, lysates ofother cells expressing the receptor, or lysates or supernatants of cellsproducing the protein as a result of DNA techniques, see below.

Generally, the purified protein will be at least about 40% pure,ordinarily at least about 50% pure, usually at least about 60% pure,typically at least about 70% pure, more typically at least about 80%pure, preferable at least about 90% pure and more preferably at leastabout 95% pure, and in particular embodiments, 97%-99% or more. Puritywill usually be on a weight basis, but can also be on a molar basis.Different assays will be applied as appropriate.

VI. Antibodies

Antibodies can be raised to the various mammalian, e.g., primate DIRS1proteins and fragments thereof, both in naturally occurring native formsand in their recombinant forms, the difference being that antibodies tothe active receptor are more likely to recognize epitopes which are onlypresent in the native conformations. Denatured antigen detection canalso be useful in, e.g., Western analysis. Anti-idiotypic antibodies arealso contemplated, which would be useful as agonists or antagonists of anatural receptor or an antibody.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the protein can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective protein, or screened for agonistic orantagonistic activity. These monoclonal antibodies will usually bindwith at least a K_(D) of about 1 mM, more usually at least about 300 μM,typically at least about 100 μM, more typically at least about 30 μM,preferably at least about 10 μM, and more preferably at least about 3 μMor better.

The antibodies, including antigen binding fragments, of this inventioncan have significant diagnostic or therapeutic value. They can be potentantagonists that bind to the receptor and inhibit binding to ligand orinhibit the ability of the receptor to elicit a biological response,e.g., act on its substrate. They also can be useful as non-neutralizingantibodies and can be coupled to toxins or radionuclides to bindproducing cells, or cells localized to the source of the interleukin.Further, these antibodies can be conjugated to drugs or othertherapeutic agents, either directly or indirectly by means of a linker.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they might bindto the receptor without inhibiting ligand or substrate binding. Asneutralizing antibodies, they can be useful in competitive bindingassays. They will also be useful in detecting or quantifying ligand.They may be used as reagents for Western blot analysis, or forimmunoprecipitation or immunopurification of the respective protein.

Protein fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. Mammalian cytokine receptors and fragments may be fused orcovalently linked to a variety of immunogens, such as keyhole limpethemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology,Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962)Specificity of Serological Reactions, Dover Publications, New York; andWilliams, et al. (1967) Methods in Immunology and Immunochemistry, Vol.1, Academic Press, New York; each of which are incorporated herein byreference, for descriptions of methods of preparing polyclonal antisera.A typical method involves hyperimmunization of an animal with anantigen. The blood of the animal is then collected shortly after therepeated immunizations and the gamma globulin is isolated.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York; and particularly in Kohler andMilstein (1975) in Nature 256: 495-497, which discusses one method ofgenerating monoclonal antibodies. Each of these references isincorporated herein by reference. Summarized briefly, this methodinvolves injecting an animal with an immunogen. The animal is thensacrificed and cells taken from its spleen, which are then fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546, each of which is hereby incorporated hereinby reference. The polypeptides and antibodies of the present inventionmay be used with or without modification, including chimeric orhumanized antibodies. Frequently, the polypeptides and antibodies willbe labeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents, teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant or chimeric immunoglobulins may beproduced, see Cabilly, U.S. Pat. No. 4,816,567; or made in transgenicmice, see, e.g., Mendez, et al. (1997) Nature Genetics 15:146-156. Thesereferences are incorporated herein by reference.

The antibodies of this invention can also be used for affinitychromatography in isolating the DIRS1 proteins or peptides. Columns canbe prepared where the antibodies are linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysatemay be passed through the column, the column washed, followed byincreasing concentrations of a mild denaturant, whereby the purifiedprotein will be released. The protein may be used to purify antibody.Conversely, the antibodies may be immunoselected or immunodepleted toprovide binding compositions of defined specificities.

The antibodies may also be used to screen expression libraries forparticular expression products. Usually the antibodies used in such aprocedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

Antibodies raised against a cytokine receptor will also be used to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of theprotein or cells which express the protein. They also will be useful asagonists or antagonists of the ligand, which may be competitiveinhibitors or substitutes for naturally occurring ligands.

A cytokine receptor protein that specifically binds to or that isspecifically immunoreactive with an antibody generated against a definedimmunogen, such as an immunogen consisting of the amino acid sequence ofSEQ ID NO: 2, is typically determined in an immunoassay. The immunoassaytypically uses a polyclonal antiserum which was raised, e.g., to aprotein of SEQ ID NO: 2. This antiserum is selected to have lowcrossreactivity against other cytokine receptor family members, e.g.,IL-12 receptor beta or gp130, preferably from the same species, and anysuch crossreactivity is removed by immunoabsorption prior to use in theimmunoassay.

In order to produce antisera for use in an immunoassay, the protein,e.g., of SEQ ID NO: 2, is isolated as described herein. For example,recombinant protein may be produced in a mammalian cell line. Anappropriate host, e.g., an inbred strain of mice such as Balb/c, isimmunized with the selected protein, typically using a standardadjuvant, such as Freund's adjuvant, and a standard mouse immunizationprotocol (see Harlow and Lane, supra). Alternatively, a syntheticpeptide derived from the sequences disclosed herein and conjugated to acarrier protein can be used an immunogen. Polyclonal sera are collectedand titered against the immunogen protein in an immunoassay, e.g., asolid phase immunoassay with the immunogen immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁴ or greater are selectedand tested for their cross reactivity against other cytokine receptorfamily members, e.g., IL-12 receptor beta and/or gp130, using acompetitive binding immunoassay such as the one described in Harlow andLane, supra, at pages 570-573. Preferably at least two cytokine receptorfamily members are used in this determination. These cytokine receptorfamily members can be produced as recombinant proteins and isolatedusing standard molecular biology and protein chemistry techniques asdescribed herein.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the protein of SEQ ID NO: 2can be immobilized to a solid support. Proteins added to the assaycompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to the proteins of IL-12receptor beta or gp130. The percent crossreactivity for the aboveproteins is calculated, using standard calculations. Those antisera withless than 10% crossreactivity with each of the proteins listed above areselected and pooled. The cross-reacting antibodies are then removed fromthe pooled antisera by immunoabsorption with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein (e.g., the DIRS1 like protein of SEQ ID NO: 2). Inorder to make this comparison, the two proteins are each assayed at awide range of concentrations and the amount of each protein required toinhibit 50% of the binding of the antisera to the immobilized protein isdetermined. If the amount of the second protein required is less thantwice the amount of the protein of the selected protein or proteins thatis required, then the second protein is said to specifically bind to anantibody generated to the immunogen.

It is understood that these cytokine receptor proteins are members of afamily of homologous proteins that comprise at least 6 so far identifiedgenes. For a particular gene product, such as the DIRS1, the term refersnot only to the amino acid sequences disclosed herein, but also to otherproteins that are allelic, non-allelic, or species variants. It is alsounderstood that the terms include nonnatural mutations introduced bydeliberate mutation using conventional recombinant technology such assingle site mutation, or by excising short sections of DNA encoding therespective proteins, or by substituting new amino acids, or adding newamino acids. Such minor alterations typically will substantiallymaintain the immunoidentity of the original molecule and/or itsbiological activity. Thus, these alterations include proteins that arespecifically immunoreactive with a designated naturally occurring DIRS1protein. The biological properties of the altered proteins can bedetermined by expressing the protein in an appropriate cell line andmeasuring the appropriate effect, e.g., upon transfected lymphocytes.Particular protein modifications considered minor would includeconservative substitution of amino acids with similar chemicalproperties, as described above for the cytokine receptor family as awhole. By aligning a protein optimally with the protein of the cytokinereceptors and by using the conventional immunoassays described herein todetermine immunoidentity, one can determine the protein compositions ofthe invention.

VII. Kits and Quantitation

Both naturally occurring and recombinant forms of the cytokine receptorlike molecules of this invention are particularly useful in kits andassay methods. For example, these methods would also be applied toscreening for binding activity, e.g., ligands for these proteins.Several methods of automating assays have been developed in recent yearsso as to permit screening of tens of thousands of compounds per year.See, e.g., a BIOMEK automated workstation, Beckman Instruments, PaloAlto, Calif., and Fodor, et al. (1991) Science 251:767-773, which isincorporated herein by reference. The latter describes means for testingbinding by a plurality of defined polymers synthesized on a solidsubstrate. The development of suitable assays to screen for a ligand oragonist/antagonist homologous proteins can be greatly facilitated by theavailability of large amounts of purified, soluble cytokine receptors inan active state such as is provided by this invention.

Purified DIRS1 can be coated directly onto plates for use in theaforementioned ligand screening techniques. However, non-neutralizingantibodies to these proteins can be used as capture antibodies toimmobilize the respective receptor on the solid phase, useful, e.g., indiagnostic uses.

This invention also contemplates use of DIRS1, fragments thereof,peptides, and their fusion products in a variety of diagnostic kits andmethods for detecting the presence of the protein or its ligand.Alternatively, or additionally, antibodies against the molecules may beincorporated into the kits and methods. Typically the kit will have acompartment containing either a DIRS1 peptide or gene segment or areagent which recognizes one or the other. Typically, recognitionreagents, in the case of peptide, would be a receptor or antibody, or inthe case of a gene segment, would usually be a hybridization probe.

A preferred kit for determining the concentration of DIRS1 in a samplewould typically comprise a labeled compound, e.g., ligand or antibody,having known binding affinity for DIRS1, a source of DIRS1 (naturallyoccurring or recombinant) as a positive control, and a means forseparating the bound from free labeled compound, for example a solidphase for immobilizing the DIRS1 in the test sample. Compartmentscontaining reagents, and instructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for mammalianDIRS1 or a peptide fragment, or receptor fragments are useful indiagnostic applications to detect the presence of elevated levels ofligand and/or its fragments. Diagnostic assays may be homogeneous(without a separation step between free reagent and antibody-antigencomplex) or heterogeneous (with a separation step). Various commercialassays exist, such as radioimmunoassay (RIA), enzyme-linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme-multipliedimmunoassay technique (EMIT), substrate-labeled fluorescent immunoassay(SLFIA) and the like. For example, unlabeled antibodies can be employedby using a second antibody which is labeled and which recognizes theantibody to a cytokine receptor or to a particular fragment thereof.These assays have also been extensively discussed in the literature.See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH.,and Coligan (ed. 1991 and periodic supplements) Current Protocols InImmunology Greene/Wiley, New York.

Anti-idiotypic antibodies may have similar use to serve as agonists orantagonists of cytokine receptors. These should be useful as therapeuticreagents under appropriate circumstances.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody, or labeled ligand is provided.This is usually in conjunction with other additives, such as buffers,stabilizers, materials necessary for signal production such assubstrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments for each useful reagent, andwill contain instructions for proper use and disposal of reagents.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium havingappropriate concentrations for performing the assay.

The aforementioned constituents of the diagnostic assays may be usedwithout modification or may be modified in a variety of ways. Forexample, labeling may be achieved by covalently or non-covalentlyjoining a moiety which directly or indirectly provides a detectablesignal. In many of these assays, a test compound, cytokine receptor, orantibodies thereto can be labeled either directly or indirectly.Possibilities for direct labeling include label groups: radiolabels suchas ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) such as peroxidase andalkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475)capable of monitoring the change in fluorescence intensity, wavelengthshift, or fluorescence polarization. Both of the patents areincorporated herein by reference. Possibilities for indirect labelinginclude biotinylation of one constituent followed by binding to avidincoupled to one of the above label groups.

There are also numerous methods of separating the bound from the freeligand, or alternatively the bound from the free test compound. Thecytokine receptor can be immobilized on various matrixes followed bywashing. Suitable matrices include plastic such as an ELISA plate,filters, and beads. Methods of immobilizing the receptor to a matrixinclude, without limitation, direct adhesion to plastic, use of acapture antibody, chemical coupling, and biotin-avidin. The last step inthis approach involves the precipitation of antibody/antigen complex byany of several methods including those utilizing, e.g., an organicsolvent such as polyethylene glycol or a salt such as ammonium sulfate.Other suitable separation techniques include, without limitation, thefluorescein antibody magnetizable particle method described in Rattle,et al. (1984) Clin. Chem. 30(9):1457-1461, and the double antibodymagnetic particle separation as described in U.S. Pat. No. 4,659,678,each of which is incorporated herein by reference.

The methods for linking protein or fragments to various labels have beenextensively reported in the literature and do not require detaileddiscussion here. Many of the techniques involve the use of activatedcarboxyl groups either through the use of carbodiimide or active estersto form peptide bonds, the formation of thioethers by reaction of amercapto group with an activated halogen such as chloroacetyl, or anactivated olefin such as maleimide, for linkage, or the like. Fusionproteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence ofan cytokine receptor. These sequences can be used as probes fordetecting levels of the respective cytokine receptor in patientssuspected of having an immunological disorder. The preparation of bothRNA and DNA nucleotide sequences, the labeling of the sequences, and thepreferred size of the sequences has received ample description anddiscussion in the literature. Normally an oligonucleotide probe shouldhave at least about 14 nucleotides, usually at least about 18nucleotides, and the polynucleotide probes may be up to severalkilobases. Various labels may be employed, most commonly radionuclides,particularly ³²P. However, other techniques may also be employed, suchas using biotin modified nucleotides for introduction into apolynucleotide. The biotin then serves as the site for binding to avidinor antibodies, which may be labeled with a wide variety of labels, suchas radionuclides, fluorescers, enzymes, or the like. Alternatively,antibodies may be employed which can recognize specific duplexes,including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, orDNA-protein duplexes. The antibodies in turn may be labeled and theassay carried out where the duplex is bound to a surface, so that uponthe formation of duplex on the surface, the presence of antibody boundto the duplex can be detected. The use of probes to the novel anti-senseRNA may be carried out in conventional techniques such as nucleic acidhybridization, plus and minus screening, recombinational probing, hybridreleased translation (HRT), and hybrid arrested translation (HART). Thisalso includes amplification techniques such as polymerase chain reaction(PCR).

Diagnostic kits which also test for the qualitative or quantitativepresence of other markers are also contemplated. Diagnosis or prognosismay depend on the combination of multiple indications used as markers.Thus, kits may test for combinations of markers. See, e.g., Viallet, etal. (1989) Progress in Growth Factor Res. 1:89-97.

VIII. Therapeutic Utility

This invention provides reagents with significant therapeutic value.See, e.g., Levitzki (1996) Curr. Opin. Cell Biol. 8:239-244. Thecytokine receptors (naturally occurring or recombinant), fragmentsthereof, mutein receptors, and antibodies, along with compoundsidentified as having binding affinity to the receptors or antibodies,should be useful in the treatment of conditions exhibiting abnormalexpression of the receptors of their ligands. Such abnormality willtypically be manifested by immunological disorders. Additionally, thisinvention should provide therapeutic value in various diseases ordisorders associated with abnormal expression or abnormal triggering ofresponse to the ligand. For example, the IL-1 ligands have beensuggested to be involved in morphologic development, e.g., dorso-ventralpolarity determination, and immune responses, particularly the primitiveinnate responses. See, e.g., Sun, et al. (1991) Eur. J. Biochem.196:247-254; and Hultmark (1994) Nature 367:116-117.

Recombinant cytokine receptors, muteins, agonist or antagonistantibodies thereto, or antibodies can be purified and then administeredto a patient. These reagents can be combined for therapeutic use withadditional active ingredients, e.g., in conventional pharmaceuticallyacceptable carriers or diluents, along with physiologically innocuousstabilizers and excipients. These combinations can be sterile, e.g.,filtered, and placed into dosage forms as by lyophilization in dosagevials or storage in stabilized aqueous preparations. This invention alsocontemplates use of antibodies or binding fragments thereof which arenot complement binding.

Ligand screening using cytokine receptor or fragments thereof can beperformed to identify molecules having binding affinity to thereceptors. Subsequent biological assays can then be utilized todetermine if a putative ligand can provide competitive binding, whichcan block intrinsic stimulating activity. Receptor fragments can be usedas a blocker or antagonist in that it blocks the activity of ligand.Likewise, a compound having intrinsic stimulating activity can activatethe receptor and is thus an agonist in that it simulates the activity ofligand, e.g., inducing signaling. This invention further contemplatesthe therapeutic use of antibodies to cytokine receptors as antagonists.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, reagent physiological life, pharmacological life, physiologicalstate of the patient, and other medicants administered. Thus, treatmentdosages should be titrated to optimize safety and efficacy. Typically,dosages used in vitro may provide useful guidance in the amounts usefulfor in situ administration of these reagents. Animal testing ofeffective doses for treatment of particular disorders will providefurther predictive indication of human dosage. Various considerationsare described, e.g., in Gilman, et al. (eds. 1990) Goodman and Gilman's:The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; andRemington's Pharmaceutical Sciences, 17th ed. (1990), Mack PublishingCo., Easton, Pa.; each of which is hereby incorporated herein byreference. Methods for administration are discussed therein and below,e.g., for oral, intravenous, intraperitoneal, or intramuscularadministration, transdermal diffusion, and others. Pharmaceuticallyacceptable carriers will include water, saline, buffers, and othercompounds described, e.g., in the Merck Index, Merck & Co., Rahway, N.J.Because of the likely high affinity binding, or turnover numbers,between a putative ligand and its receptors, low dosages of thesereagents would be initially expected to be effective. And the signalingpathway suggests extremely low amounts of ligand may have effect. Thus,dosage ranges would ordinarily be expected to be in amounts lower than 1mM concentrations, typically less than about 10 μM concentrations,usually less than about 100 nM, preferably less than about 10 pM(picomolar), and most preferably less than about 1 fM (femtomolar), withan appropriate carrier. Slow release formulations, or slow releaseapparatus will often be utilized for continuous administration.

Cytokine receptors, fragments thereof, and antibodies or its fragments,antagonists, and agonists, may be administered directly to the host tobe treated or, depending on the size of the compounds, it may bedesirable to conjugate them to carrier proteins such as ovalbumin orserum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationscomprise at least one active ingredient, as defined above, together withone or more acceptable carriers thereof. Each carrier must be bothpharmaceutically and physiologically acceptable in the sense of beingcompatible with the other ingredients and not injurious to the patient.Formulations include those suitable for oral, rectal, nasal, orparenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by methods well knownin the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990)Goodman andGilman's: The Pharmacological Bases of Therapeutics, 8th Ed., PergamonPress; and Remington's Pharmaceutical Sciences, 17th ed. (1990), MackPublishing Co., Easton, Pa.; Avis, et al. (eds. 1993) PharmaceuticalDosage Forms: Parenteral Medications Dekker, NY; Lieberman, et al. (eds.1990) Pharmaceutical Dosage Forms: Tablets Dekker, NY; and Lieberman, etal. (eds. 1990) Pharmaceutical Dosage Forms Disperse Systems Dekker, NY.The therapy of this invention may be combined with or used inassociation with other therapeutic agents, particularly agonists orantagonists of other cytokine receptor family members.

IX. Screening

Drug screening using DIRS1 or fragments thereof can be performed toidentify compounds having binding affinity to the receptor subunit,including isolation of associated components. Subsequent biologicalassays can then be utilized to determine if the compound has intrinsicstimulating activity and is therefore a blocker or antagonist in that itblocks the activity of the ligand. Likewise, a compound having intrinsicstimulating activity can activate the receptor and is thus an agonist inthat it simulates the activity of a cytokine ligand. This inventionfurther contemplates the therapeutic use of antibodies to the receptoras cytokine agonists or antagonists.

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing the DIRS1. Cells may be isolated which express a receptor inisolation from other functional receptors. Such cells, either in viableor fixed form, can be used for standard ligand/receptor binding assays.See also, Parce, et al. (1989) Science 246:243-247; and Owicki, et al.(1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses. Competitive assays areparticularly useful, where the cells (source of putative ligand) arecontacted and incubated with a labeled receptor or antibody having knownbinding affinity to the ligand, such as ¹²⁵I-antibody, and a test samplewhose binding affinity to the binding composition is being measured. Thebound and free labeled binding compositions are then separated to assessthe degree of ligand binding. The amount of test compound bound isinversely proportional to the amount of labeled receptor binding to theknown source. Any one of numerous techniques can be used to separatebound from free ligand to assess the degree of ligand binding. Thisseparation step could typically involve a procedure such as adhesion tofilters followed by washing, adhesion to plastic followed by washing, orcentrifugation of the cell membranes. Viable cells could also be used toscreen for the effects of drugs on cytokine mediated functions, e.g.,second messenger levels, i.e., Ca⁺⁺; cell proliferation; inositolphosphate pool changes; and others. Some detection methods allow forelimination of a separation step, e.g., a proximity sensitive detectionsystem. Calcium sensitive dyes will be useful for detecting Ca⁺⁺ levels,with a fluorimeter or a fluorescence cell sorting apparatus.

X. Ligands

The descriptions of the DIRS1 herein provide means to identify ligands,as described above. Such ligand should bind specifically to therespective receptor with reasonably high affinity. Various constructsare made available which allow either labeling of the receptor to detectits ligand. For example, directly labeling cytokine receptor, fusingonto it markers for secondary labeling, e.g., FLAG or other epitopetags, etc., will allow detection of receptor. This can be histological,as an affinity method for biochemical purification, or labeling orselection in an expression cloning approach. A two-hybrid selectionsystem may also be applied making appropriate constructs with theavailable cytokine receptor sequences. See, e.g., Fields and Song (1989)Nature 340:245-246.

Generally, descriptions of cytokine receptors will be analogouslyapplicable to individual specific embodiments directed to DIRS1 reagentsand compositions.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

EXAMPLES I. General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene/Wiley, New York. Methods forprotein purification include such methods as ammonium sulfateprecipitation, column chromatography, electrophoresis, centrifugation,crystallization, and others. See, e.g., Ausubel, et al. (1987 andperiodic supplements); Coligan, et al. (ed. 1996) and periodicsupplements, Current Protocols In Protein Science Greene/Wiley, NewYork; Deutscher (1990) “Guide to Protein Purification” in Methods inEnzymology, vol. 182, and other volumes in this series; andmanufacturer's literature on use of protein purification products, e.g.,Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combinationwith recombinant techniques allow fusion to appropriate segments, e.g.,to a FLAG sequence or an equivalent which can be fused via aprotease-removable sequence. See, e.g., Hochuli (1989) ChemischeIndustrie 12:69-70; Hochuli (1990) “Purification of Recombinant Proteinswith Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering,Principle and Methods 12:87-98, Plenum Press, N.Y.; and Crowe, et al.(1992) QIAexpress: The High Level Expression & Protein PurificationSystem QUIAGEN, Inc., Chatsworth, Calif.

Computer sequence analysis is performed, e.g., using available softwareprograms, including those from the GCG (U. Wisconsin) and GenBanksources. Public sequence databases were also used, e.g., from GenBankand others.

Many techniques applicable to IL-10 or IL-12 receptors may be applied tothe DIRS1, as described, e.g., in U.S. Ser. No. 08/110,683 (IL-10receptor), which is incorporated herein by reference.

II. Computational Analysis

Human sequences related to cytokine receptors were identified fromgenomic sequence database using, e.g., the BLAST server (Altschul, etal. (1994) Nature Genet. 6:119-129). Standard analysis programs may beused to evaluate structure, e.g., PHD (Rost and Sander (1994) Proteins19:55-72) and DSC (King and Sternberg (1996) Protein Sci. 5:2298-2310).Standard comparison software includes, e.g., Altschul, et al. (1990) J.Mol. Biol. 215:403-10; Waterman (1995) Introduction to ComputationalBiology: Maps, Sequences, and Genomes Chapman & Hall; Lander andWaterman (eds. 1995) Calculating the Secrets of Life: Applications ofthe Mathematical Sciences in Molecular Biology National Academy Press;and Speed and Waterman (eds. 1996) Genetic Mapping and DNA Sequencing(Ima Volumes in Mathematics and Its Applications, Vol 81) SpringerVerlag.

III. Cloning of Full-Length DIRS cDNAs; Chromosomal Localization

PCR primers derived from the DIRS sequences are used to probe a humancDNA library. Full length cDNAs for primate, rodent, or other speciesDIRS1 are cloned, e.g., by DNA hybridization screening of λgt10 phage.PCR reactions are conducted using T. aquaticus Taqplus DNA polymerase(Stratagene) under appropriate conditions.

Chromosome spreads are prepared. In situ hybridization is performed onchromosome preparations obtained from phytohemagglutinin-stimulatedhuman lymphocytes cultured for 72 h. 5-bromodeoxyuridine was added forthe final seven hours of culture (60 μg/ml of medium), to ensure aposthybridization chromosomal banding of good quality.

A PCR fragment, amplified with the help of primers, is cloned into anappropriate vector. The vector is labeled by nick-translation with ³H.The radiolabeled probe is hybridized to metaphase spreads at finalconcentration of 200 ng/ml of hybridization solution as described inMattei, et al. (1985) Hum. Genet. 69:327-331.

After coating with nuclear track emulsion (KODAK NTB₂), slides areexposed. To avoid any slipping of silver grains during the bandingprocedure, chromosome spreads are first stained with buffered Giemsasolution and metaphase photographed. R-banding is then performed by thefluorochrome-photolysis-Giemsa (FPG) method and metaphasesrephotographed before analysis. Alternatively, mapped sequence tags maybe searched in a database.

Similar appropriate methods are used for other species.

IV. Localization of DIRS1 or DIRS2 mRNA

Human multiple tissue (Cat# 1, 2) and cancer cell line blots (Cat#7757-1), containing approximately 2 μg of poly(A)⁺ RNA per lane, arepurchased from Clontech (Palo Alto, Calif.). Probes are radiolabeledwith [α-³²P] dATP, e.g., using the Amersham Rediprime random primerlabeling kit (RPN1633). Prehybridization and hybridizations areperformed at 65° C. in 0.5 M Na₂HPO₄, 7% SDS, 0.5 M EDTA (pH 8.0). Highstringency washes are conducted, e.g., at 65° C. with two initial washesin 2×SSC, 0.1% SDS for 40 min followed by a subsequent wash in 0.1×SSC,0.1% SDS for 20 min. Membranes are then exposed at −70° C. to X-Ray film(Kodak) in the presence of intensifying screens. More detailed studiesby cDNA library Southerns are performed with selected human DIRS1 clonesto examine their expression in hemopoietic or other cell subsets.

Alternatively, two appropriate primers are selected from Table 1 or 2.RT-PCR is used on an appropriate mRNA sample selected for the presenceof message to produce a cDNA, e.g., a sample which expresses the gene.

Full length clones may be isolated by hybridization of cDNA librariesfrom appropriate tissues pre-selected by PCR signal. Northern blots canbe performed.

Message for genes encoding DIRS1 will be assayed by appropriatetechnology, e.g., PCR, immunoassay, hybridization, or otherwise. Tissueand organ cDNA preparations are available, e.g., from Clontech, MountainView, Calif. Identification of sources of natural expression are useful,as described. And the identification of functional receptor subunitpairings will allow for prediction of what cells express the combinationof receptor subunits which will result in a physiological responsivenessto each of the cytokine ligands.

For mouse distribution, e.g., Southern Analysis can be performed: DNA (5μg) from a primary amplified cDNA library was digested with appropriaterestriction enzymes to release the inserts, run on a 1% agarose gel andtransferred to a nylon membrane (Schleicher and Schuell, Keene, N.H.).

Samples for mouse mRNA isolation may include: resting mouse fibroblasticL cell line (C200); Braf:ER (Braf fusion to estrogen receptor)transfected cells, control (C201); T cells, TH1 polarized (Mel14 bright,CD4+ cells from spleen, polarized for 7 days with IFN-γ and anti IL-4;T200); T cells, TH2 polarized (Mel14 bright, CD4+ cells from spleen,polarized for 7 days with IL-4 and anti-IFN-γ; T201); T cells, highlyTH1 polarized (see Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367;activated with anti-CD3 for 2, 6, 16 h pooled; T202); T cells, highlyTH2 polarized (see Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367;activated with anti-CD3 for 2, 6, 16 h pooled; T203); CD44− CD25+ pre Tcells, sorted from thymus (T204); TH1 T cell clone D1.1, resting for 3weeks after last stimulation with antigen (T205); TH1 T cell clone D1.1,10 μg/ml ConA stimulated 15 h (T206); TH2 T cell clone CDC35, restingfor 3 weeks after last stimulation with antigen (T207); TH2 T cell cloneCDC35, 10 μg/ml ConA stimulated 15 h (T208); Mel14+ naive T cells fromspleen, resting (T209); Mel14+ T cells, polarized to Th1 withIFN-γ/IL-12/anti-IL-4 for 6, 12, 24 h pooled (T210); Mel14+ T cells,polarized to Th2 with IL-4/anti-IFN-γ for 6, 13, 24 h pooled (T211);unstimulated mature B cell leukemia cell line A20 (B200); unstimulated Bcell line CH12 (B201); unstimulated large B cells from spleen (B202); Bcells from total spleen, LPS activated (B203); metrizamide enricheddendritic cells from spleen, resting (D200); dendritic cells from bonemarrow, resting (D201); monocyte cell line RAW 264.7 activated with LPS4 h (M200); bone-marrow macrophages derived with GM and M-CSF (M201);macrophage cell line J774, resting (M202); macrophage cell lineJ774+LPS+anti-IL-10 at 0.5, 1, 3, 6, 12 h pooled (M203); macrophage cellline J774+LPS+IL-10 at 0.5, 1, 3, 5, 12 h pooled (M204); aerosolchallenged mouse lung tissue, Th2 primers, aerosol OVA challenge 7, 14,23 h pooled (see Garlisi, et al. (1995) Clinical Immunology andImmunopathology 75:75-83; X206); Nippostrongulus-infected lung tissue(see Coffman, et al. (1989) Science 245:308-310; X200); total adultlung, normal (O200); total lung, rag-1 (see Schwarz, et al. (1993)Immunodeficiency 4:249-252; 0205); IL-10 K.O. spleen (see Kuhn, et al.(1991) Cell 75:263-274; X201); total adult spleen, normal (O201); totalspleen, rag-1 (O207); IL-10 K.O. Peyer's patches (O202); total Peyer'spatches, normal (O210); IL-10 K.O. mesenteric lymph nodes (X203); totalmesenteric lymph nodes, normal (O211); IL-10 K.O. colon (X203); totalcolon, normal (O212); NOD mouse pancreas (see Makino, et al. (1980)Jikken Dobutsu 29:1-13; X205); total thymus, rag-1 (O208); total kidney,rag-1 (O209); total heart, rag-1 (O202); total brain, rag-1 (O203);total testes, rag-1 (O204); total liver, rag-1 (O206); rat normal jointtissue (O300); and rat arthritic joint tissue (X300).

Samples for human mRNA isolation may include: peripheral bloodmononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells),resting (T100); peripheral blood mononuclear cells, activated withanti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72, resting(T102); T cell, TH0 clone Mot 72, activated with anti-CD28 and anti-CD3for 3, 6, 12 h pooled (T103); T cell, TH0 clone Mot 72, anergic treatedwith specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 cloneHY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06,anergic treated with specific peptide for 2, 6, 12 h pooled (T109); Tcell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); Tcells CD4+CD45RO− T cells polarized 27 days in anti-CD28, IL-4, and antiIFN-γ, TH2 polarized, activated with anti-CD3 and anti-CD28 4 h (T116);T cell tumor lines Jurkat and Hut78, resting (T117); T cell clones,pooled AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118);T cell random γδ T cell clones, resting (T119); Splenocytes, resting(B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); B cellEBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting (B102);B cell line JY, activated with PMA and ionomycin for 1, 6 h pooled(B103); NK 20 clones pooled, resting (K100); NK 20 clones pooled,activated with PMA and ionomycin for 6 h (K101); NKL clone, derived fromperipheral blood of LGL leukemia patient, IL-2 treated (K106); NKcytotoxic clone 640-A30-1, resting (K107); hematopoietic precursor lineTF1, activated with PMA and ionomycin for 1, 6 h pooled (C100); U937premonocytic line, resting (M100); U937 premonocytic line, activatedwith PMA and ionomycin for 1, 6 h pooled (M101); elutriated monocytes,activated with LPS, IFNγ, anti-IL-10 for 1, 2, 6, 12, 24 h pooled(M102); elutriated monocytes, activated with LPS, IFNγ, IL-10 for 1, 2,6, 12, 24 h pooled (M103); elutriated monocytes, activated with LPS,IFNγ, anti-IL-10 for 4, 16 h pooled (M106); elutriated monocytes,activated with LPS, IFNγ, IL-10 for 4, 16 h pooled (M107); elutriatedmonocytes, activated LPS for 1 h (M108); elutriated monocytes, activatedLPS for 6 h (M109); DC 70% CD1a+, from CD34+ GM-CSF, TNFα 12 days,resting (D101); DC 70% CD1a+, from CD34+ GM-CSF, TNFα 12 days, activatedwith PMA and ionomycin for 1 hr (D102); DC 70% CD1a+, from CD34+ GM-CSF,TNFα 12 days, activated with PMA and ionomycin for 6 hr (D103); DC 95%CD1a+, from CD34+ GM-CSF, TNFα 12 days FACS sorted, activated with PMAand ionomycin for 1, 6 h pooled (D104); DC 95% CD14+, ex CD34+ GM-CSF,TNFα 12 days FACS sorted, activated with PMA and ionomycin 1, 6 hrpooled (D105); DC CD1a+ CD86+, from CD34+ GM-CSF, TNFα 12 days FACSsorted, activated with PMA and ionomycin for 1, 6 h pooled (D106); DCfrom monocytes GM-CSF, IL-4 5 days, resting (D107); DC from monocytesGM-CSF, IL-4 5 days, resting (D108); DC from monocytes GM-CSF, IL-4 5days, activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF,IL-4 5 days, activated TNFα, monocyte supe for 4, 16 h pooled (D110);leiomyoma L11 benign tumor (X101); normal myometrium M5 (O115);malignant leiomyosarcoma GS1 (X103); lung fibroblast sarcoma line MRC5,activated with PMA and ionomycin for 1, 6 h pooled (C101); kidneyepithelial carcinoma cell line CHA, activated with PMA and ionomycin for1, 6 h pooled (C102); kidney fetal 28 wk male (O100); lung fetal 28 wkmale (O101); liver fetal 28 wk male (O102); heart fetal 28 wk male(O103); brain fetal 28 wk male (O104); gallbladder fetal 28 wk male(O106); small intestine fetal 28 wk male (O107); adipose tissue fetal 28wk male (O108); ovary fetal 25 wk female (O109); uterus fetal 25 wkfemale (O110); testes fetal 28 wk male (O111); spleen fetal 28 wk male(O112); adult placenta 28 wk (O113); and tonsil inflamed, from 12 yearold (X100).

With a cDNA Southern, the human DIRS1 was found in LPS activateddendritic cells (“DC LPS”); monokine activated dendritic cells (“DCmix”); normal skin; Psoriasis skin; inflamed tonsil; fetal liver; fetalsmall intestine; fetal ovary; resting “70% dendritic cells”; 6 hractivated 70% dendritic cells; and LPS activated monocytes. A signal wasalso detected in normal monkey lung and Ascaris-challenged monkey lung(24 h), which indicates cross species hybridization. The followinglibraries had weaker expression of DIRS1: smoker lung pool; fetal spleenCD4+ T cells (TH2 polarized); gamma delta T cells; activatedsplenocytes; and B cells.

HOFNy28 (DIRS2) is expressed in U937 (a premonocytic cell line) cells,both resting and activated; activated A549 cells (epithelial cells,IL-1β activated); fetal uterus; fetal testes; and fetal spleen. Thisdata is from PCR on these cDNA libraries followed by Southernhybridization.

Similar samples may isolated in other species for evaluation.

V. Cloning of Species Counterparts of DIRS1 or DIRS2

Various strategies are used to obtain species counterparts of, e.g., theDIRS1, preferably from other primates or rodents. One method is by crosshybridization using closely related species DNA probes. It may be usefulto go into evolutionarily similar species as intermediate steps. Anothermethod is by using specific PCR primers based on the identification ofblocks of similarity or difference between genes, e.g., areas of highlyconserved or nonconserved polypeptide or nucleotide sequence. Databasesequence searches may also identify species counterparts.

VI. Production of Mammalian DIRS1 or DIRS2 Protein

An appropriate, e.g., GST, fusion construct is engineered forexpression, e.g., in E. coli. For example, a mouse IGIF pGex plasmid isconstructed and transformed into E. coli. Freshly transformed cells aregrown, e.g., in LB medium containing 50 μg/ml ampicillin and inducedwith IPTG (Sigma, St. Louis, Mo.). After overnight induction, thebacteria are harvested and the pellets containing the DIRS1 protein areisolated. The pellets are homogenized, e.g., in TE buffer (50 mMTris-base pH 8.0, 10 mM EDTA and 2 mM pefabloc) in 2 liters. Thismaterial is passed through a microfluidizer (Microfluidics, Newton,Mass.) three times. The fluidized supernatant is spun down on a SorvallGS-3 rotor for 1 h at 13,000 rpm. The resulting supernatant containingthe cytokine receptor protein is filtered and passed over aglutathione-SEPHAROSE column equilibrated in 50 mM Tris-base pH 8.0. Thefractions containing the DIRS1-GST fusion protein are pooled andcleaved, e.g., with thrombin (Enzyme Research Laboratories, Inc., SouthBend, Ind.). The cleaved pool is then passed over a Q-SEPHAROSE columnequilibrated in 50 mM Tris-base. Fractions containing DIRS1 are pooledand diluted in cold distilled H₂O, to lower the conductivity, and passedback over a fresh Q-Sepharose column, alone or in succession with animmunoaffinity antibody column. Fractions containing the DIRS1 proteinare pooled, aliquoted, and stored in the −70° C. freezer.

Comparison of the CD spectrum with cytokine receptor protein may suggestthat the protein is correctly folded. See Hazuda, et al. (1969) J. Biol.Chem. 264:1689-1693.

VII. Determining Physiological Forms of Receptors

The cellular forms of receptors for ligands can be tested with thevarious ligands and receptor subunits provided, e.g., IL-10 relatedsequences. In particular, multiple cytokine receptor like ligands havebeen identified, see, e.g., U.S. Ser. No. 60/027,368, 08/934,959, and08/842,659, which are incorporated herein by reference.

Cotransformation of the DIRS1 with putative other receptor subunit genesmay be performed. In particular, the DSRS1 is suggested to be a secondreceptor subunit needed for functional receptor signaling. Such cellsmay be used to screen putative cytokine ligands, such as the DIL-30, forsignaling. A cell proliferation assay may be used.

In addition, it has been known that many cytokine receptors function asheterodimers. The IL-1α and IL-1β ligands bind an IL-1R1 as the primaryreceptor and this complex then forms a high affinity receptor complexwith the IL-1R3. As indicated above, the sequence similarity to IL-12receptor subunits suggests functional similarity of the functionalreceptor, e.g., a soluble alpha subunit, and transmembrane beta subunit.

These subunit combinations can be tested now with the provided reagents.In particular, appropriate constructs can be made for transformation ortransfection of subunits into cells. Constructs for the alpha chains,e.g., DSRS1 forms, can be made. Likewise for the beta subunit DIRS1.Combinatorial transfections of transformations can make cells expressingdefined subunits, which can be tested for response to the predictedligands. Appropriate cell types can be used, e.g., 293 T cells, with,e.g., an NFκb reporter construct.

Biological assays will generally be directed to the ligand bindingfeature of the protein or to the kinase/phosphatase activity of thereceptor. The activity will typically be reversible, as are many otherenzyme reactions, and may mediate phosphatase or phosphorylaseactivities, which activities are easily measured by standard procedures.See, e.g., Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols.I and II, Academic Press, San Diego, Calif.; Hanks, et al. (1991) Meth.Enzymol. 200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990)Cell 61:743-752; Pines, et al. (1991) Cold Spring Harbor Symp. Quant.Biol. 56:449-463; and Parker, et al. (1993) Nature 363:736-738.

The family of cytokines contains molecules which are important mediatorsof hematopoiesis or inflammatory disease. See, e.g., Thomson (ed. 1994)The Cytokine Handbook Academic Press, San Diego; and Dinarello (1996)Blood 87:2095-2147.

VIII. Antibodies Specific for DIRS1 or DIRS2

Inbred Balb/c mice are immunized intraperitoneally with recombinantforms of the protein, e.g., purified DIRS1 or stable transfected N1H-3T3cells. Animals are boosted at appropriate time points with protein, withor without additional adjuvant, to further stimulate antibodyproduction. Serum is collected, or hybridomas produced with harvestedspleens.

Alternatively, Balb/c mice are immunized with cells transformed with thegene or fragments thereof, either endogenous or exogenous cells, or withisolated membranes enriched for expression of the antigen. Serum iscollected at the appropriate time, typically after numerous furtheradministrations. Various gene therapy techniques may be useful, e.g., inproducing protein in situ, for generating an immune response. Serum maybe immunoselected or depleted to prepare substantially purifiedantibodies of defined specificity and high affinity. Preparations whichspecifically bind particular segments or defined epitopes may be made.

Monoclonal antibodies may be made. For example, splenocytes are fusedwith an appropriate fusion partner and hybridomas are selected in growthmedium by standard procedures. Hybridoma supernatants are screened forthe presence of antibodies which bind to the DIRS1, e.g., by ELISA orother assay. Antibodies which specifically recognize specific DIRS1embodiments may also be selected or prepared.

In another method, synthetic peptides or purified protein are presentedto an immune system to generate monoclonal or polyclonal antibodies.See, e.g., Coligan (ed. 1991) Current Protocols in ImmunologyWiley/Greene; and Harlow and Lane (1989) Antibodies: A Laboratory ManualCold Spring Harbor Press. In appropriate situations, the binding reagentis either labeled as described above, e.g., fluorescence or otherwise,or immobilized to a substrate for panning methods. Nucleic acids mayalso be introduced into cells in an animal to produce the antigen, whichserves to elicit an immune response. See, e.g., Wang, et al. (1993)Proc. Nat'l. Acad. Sci. 90:4156-4160; Barry, et al. (1994) BioTechniques16:616-619; and Xiang, et al. (1995) Immunity 2: 129-135.

Moreover, antibodies which may be useful to determine the combination ofthe DIRS1 with a functional alpha subunit may be generated. Thus, e.g.,epitopes characteristic of a particular functional alpha/betacombination may be identified with appropriate antibodies.

IX. Production of Fusion Proteins with DIRS1 or DIRS2

Various fusion constructs are made with DIRS1 or DIRS2. A portion of theappropriate gene is fused to an epitope tag, e.g., a FLAG tag, or to atwo hybrid system construct. See, e.g., Fields and Song (1989) Nature340:245-246.

The epitope tag may be used in an expression cloning procedure withdetection with anti-FLAG antibodies to detect a binding partner, e.g.,ligand for the respective cytokine receptor. The two hybrid system mayalso be used to isolate proteins which specifically bind to DIRS1.

X. Structure Activity Relationship

Information on the criticality of particular residues is determinedusing standard procedures and analysis. Standard mutagenesis analysis isperformed, e.g., by generating many different variants at determinedpositions, e.g., at the positions identified above, and evaluatingbiological activities of the variants. This may be performed to theextent of determining positions which modify activity, or to focus onspecific positions to determine the residues which can be substituted toeither retain, block, or modulate biological activity.

Alternatively, analysis of natural variants can indicate what positionstolerate natural mutations. This may result from populational analysisof variation among individuals, or across strains or species. Samplesfrom selected individuals are analyzed, e.g., by PCR analysis andsequencing. This allows evaluation of population polymorphisms.

XI. Isolation of a Ligand for DIRS1 or DIRS2

A cytokine receptor can be used as a specific binding reagent toidentify its binding partner, by taking advantage of its specificity ofbinding, much like an antibody would be used. Typically, the bindingreceptor is a heterodimer of receptor subunits. A binding reagent iseither labeled as described above, e.g., fluorescence or otherwise, orimmobilized to a substrate for panning methods.

The binding composition is used to screen an expression library madefrom a cell line which expresses a binding partner, i.e., ligand,preferably membrane associated. Standard staining techniques are used todetect or sort surface expressed ligand, or surface expressingtransformed cells are screened by panning. Screening of intracellularexpression is performed by various staining or immunofluorescenceprocedures. See also McMahan, et al. (1991) EMBO J. 10:2821-2832.

For example, on day 0, precoat 2-chamber permanox slides with 1 ml perchamber of fibronectin, 10 ng/ml in PBS, for 30 min at room temperature.Rinse once with PBS. Then plate COS cells at 2-3×10⁵ cells per chamberin 1.5 ml of growth media. Incubate overnight at 37° C.

On day 1 for each sample, prepare 0.5 ml of a solution of 66 μg/mlDEAE-dextran, 66 μM chloroquine, and 4 μg DNA in serum free DME. Foreach set, a positive control is prepared, e.g., of DIRS1-FLAG cDNA at 1and 1/200 dilution, and a negative mock. Rinse cells with serum freeDME. Add the DNA solution and incubate 5 hr at 37° C. Remove the mediumand add 0.5 ml 10% DMSO in DME for 2.5 min. Remove and wash once withDME. Add 1.5 ml growth medium and incubate overnight.

On day 2, change the medium. On days 3 or 4, the cells are fixed andstained. Rinse the cells twice with Hank's Buffered Saline Solution(HBSS) and fix in 4% paraformaldehyde (PFA)/glucose for 5 min. Wash 3×with HBSS. The slides may be stored at −80° C. after all liquid isremoved. For each chamber, 0.5 ml incubations are performed as follows.Add HBSS/saponin (0.1%) with 32 μl/ml of 1 M NaN₃ for 20 min. Cells arethen washed with HBSS/saponin 1×. Add appropriate DIRS1 orDIRS1/antibody complex to cells and incubate for 30 min. Wash cellstwice with HBSS/saponin. If appropriate, add first antibody for 30 min.Add second antibody, e.g., Vector anti-mouse antibody, at 1/200dilution, and incubate for 30 min. Prepare ELISA solution, e.g., VectorElite ABC horseradish peroxidase solution, and preincubate for 30 min.Use, e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin)per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRPsolution and incubate for 30 min. Wash cells twice with HBSS, secondwash for 2 min, which closes cells. Then add Vector diaminobenzoic acid(DAB) for 5 to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2drops of H₂O₂ per 5 ml of glass distilled water. Carefully removechamber and rinse slide in water. Air dry for a few minutes, then add 1drop of Crystal Mount and a cover slip. Bake for 5 min at 85-90° C.

Evaluate positive staining of pools and progressively subclone toisolation of single genes responsible for the binding.

Alternatively, receptor reagents are used to affinity purify or sort outcells expressing a putative ligand. See, e.g., Sambrook, et al. orAusubel, et al.

Another strategy is to screen for a membrane bound receptor by panning.The receptor cDNA is constructed as described above. The ligand can beimmobilized and used to immobilize expressing cells. Immobilization maybe achieved by use of appropriate antibodies which recognize, e.g., aFLAG sequence of a DIRS1 fusion construct, or by use of antibodiesraised against the first antibodies. Recursive cycles of selection andamplification lead to enrichment of appropriate clones and eventualisolation of receptor expressing clones.

Phage expression libraries can be screened by mammalian DIRS1.Appropriate label techniques, e.g., anti-FLAG antibodies, will allowspecific labeling of appropriate clones.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

1. A composition of matter selected from: a) a substantially pure orrecombinant DIRS1 polypeptide comprising at least three distinctnonoverlapping segments of at least four amino acids identical tosegments of SEQ ID NO: 2; b) a substantially pure or recombinant DIRS1polypeptide comprising at least two distinct nonoverlapping segments ofat least five amino acids identical to segments of SEQ ID NO: 2; c) anatural sequence DIRS1 comprising mature SEQ ID NO: 2; d) a fusionpolypeptide comprising DIRS1 sequence; e) a substantially pure orrecombinant DIRS2 polypeptide comprising at least three distinctnonoverlapping segments of at least ten amino acids identical tosegments of SEQ ID NO: 4; f) a substantially pure or recombinant DIRS2polypeptide comprising at least two distinct nonoverlapping segments ofat least eleven amino acids identical to segments of SEQ ID NO: 4; g) anatural sequence DIRS2 comprising SEQ ID NO: 4; or h) a fusionpolypeptide comprising DIRS2 sequence.
 2. The substantially pure orisolated antigenic: A) DIRS1 polypeptide of claim 1, wherein saiddistinct nonoverlapping segments of identity: a) include one of at leasteight amino acids; b) include one of at least four amino acids and asecond of at least five amino acids; c) include at least three segmentsof at least four, five, and six amino acids, or d) include one of atleast twelve amino acids; or B) DIRS2 polypeptide of claim 1, whereinsaid distinct nonoverlapping segments of identity: a) include one of atleast thirteen amino acids; b) include one of at least eleven aminoacids and a second of at least thirteen amino acids; c) include at leastthree segments of at least ten, eleven, and twelve amino acids; or d)include one of at least twenty-five amino acids.
 3. The composition ofmatter of claim 1, wherein said: a) DIRS1 polypeptide: i) comprises amature sequence of Table 1; ii) is an unglycosylated form of DIRS1; iii)is from a primate, such as a human; iv) comprises at least seventeenamino acids of SEQ ID NO: 2; v) exhibits at least four nonoverlappingsegments of at least seven amino acids of SEQ ID NO: 2; vi) is a naturalallelic variant of DIRS1; vii) has a length at least about 30 aminoacids; viii) exhibits at least two non-overlapping epitopes which arespecific for a primate DIRS1; ix) is glycosylated; x) has a molecularweight of at least 30 kD with natural glycosylation; xi) is a syntheticpolypeptide; xii) is attached to a solid substrate; xiii) is conjugatedto another chemical moiety; xiv) is a 5-fold or less substitution fromnatural sequence; or xv) is a deletion or insertion variant from anatural sequence; or b) DIRS2 polypeptide: i) comprises a maturesequence of Table 2; ii) is an unglycosylated form of DIRS2; iii) isfrom a primate, such as a human; iv) comprises at thirty-five aminoacids of SEQ ID NO: 4; v) exhibits at least four nonoverlapping segmentsof at least twelve amino acids of SEQ ID NO: 4; vi) is a natural allelicvariant of DIRS2; vii) has a length at least about 30 amino acids; viii)exhibits at least two non-overlapping epitopes which are specific for aprimate DIRS2; ix) is glycosylated; x) has a molecular weight of atleast 30 kD with natural glycosylation; xi) is a synthetic polypeptide;xii) is attached to a solid substrate; xiii) is conjugated to anotherchemical moiety; xiv) is a 5-fold or less substitution from naturalsequence; or xv) is a deletion or insertion variant from a naturalsequence.
 4. A composition comprising: a) a substantially pure DIRS1 andanother Interferon Receptor family member; b) a substantially pure DIRS2and another Interferon Receptor family member; c) a sterile DIRS1polypeptide of claim 1; d) a sterile DIRS2 polypeptide of claim 1; e)said DIRS1 polypeptide of claim 1 and a carrier, wherein said carrieris: i) an aqueous compound, including water, saline, and/or buffer;and/or ii) formulated for oral, rectal, nasal, topical, or parenteraladministration; or f) said DIRS2 polypeptide of claim 1 and a carrier,wherein said carrier is: i) an aqueous compound, including water,saline, and/or buffer; and/or ii) formulated for oral, rectal, nasal,topical, or parenteral administration.
 5. The fusion polypeptide ofclaim 1, comprising: a) mature protein sequence of Table 1; b) matureprotein sequence of Table 2; c) a detection or purification tag,including a FLAG, His 6, or Ig sequence; or d) sequence of anotherinterferon receptor protein.
 6. A kit comprising a polypeptide of claim1, and: a) a compartment comprising said protein or polypeptide; or b)instructions for use or disposal of reagents in said kit.
 7. A bindingcompound comprising an antigen binding site from an antibody, whichspecifically binds to a natural: A) DIRS1 polypeptide of claim 1,wherein: a) said binding compound is in a container; b) said DIRS1polypeptide is from a human; c) said binding compound is an Fv, Fab, orFab2 fragment; d) said binding compound is conjugated to anotherchemical moiety; or e) said antibody: i) is raised against a peptidesequence of a mature polypeptide of Table 1; ii) is raised against amature DIRS1; iii) is raised to a purified human DIRS1; iv) isimmunoselected; v) is a polyclonal antibody; vi) binds to a denaturedDIRS1; vii) exhibits a Kd to antigen of at least 30 μM; viii) isattached to a solid substrate, including a bead or plastic membrane; ix)is in a sterile composition; or x) is detectably labeled, including aradioactive or fluorescent label; or B) DIRS2 polypeptide of claim 1,wherein: a) said binding compound is in a container; b) said DIRS2protein is from a human; c) said binding compound is an Fv, Fab, or Fab2fragment; d) said binding compound is conjugated to another chemicalmoiety; or e) said antibody: i) is raised against a peptide sequence ofa mature polypeptide of Table 2; ii) is raised against a mature DIRS2;iii) is raised to a purified human DIRS2; iv) is immunoselected; v) is apolyclonal antibody; vi) binds to a denatured DIRS2; vii) exhibits a Kdto antigen of at least 30 μM; viii) is attached to a solid substrate,including a bead or plastic membrane; ix) is in a sterile composition;or x) is detectably labeled, including a radioactive or fluorescentlabel.
 8. A kit comprising said binding compound of claim 7, and: a) acompartment comprising said binding compound; or b) instructions for useor disposal of reagents in said kit.
 9. A method of producing anantigen:antibody complex, comprising contacting under appropriateconditions: a) a primate DIRS1 polypeptide with an antibody of claim 7A;or b) a primate DIRS2 polypeptide with an antibody of claim 7B; therebyallowing said complex to form.
 10. The method of claim 9, wherein: a)said complex is purified from other interferon receptors; b) saidcomplex is purified from other antibody; c) said contacting is with asample comprising an interferon; d) said contacting allows quantitativedetection of said antigen; e) said contacting is with a samplecomprising said antibody; or f) said contacting allows quantitativedetection of said antibody.
 11. A composition comprising: a) a sterilebinding compound of claim 7; or b) said binding compound of claim 7 anda carrier, wherein said carrier is: i) an aqueous compound, includingwater, saline, and/or buffer; and/or ii) formulated for oral, rectal,nasal, topical, or parenteral administration.
 12. An isolated orrecombinant nucleic acid encoding said: A) DIRS1 polypeptide of claim 1,wherein said: a) DIRS1 is from a human; or b) said nucleic acid: i)encodes an antigenic peptide sequence of Table 1; ii) encodes aplurality of antigenic peptide sequences of Table 1; iii) exhibitsidentity over at least thirteen nucleotides to a natural cDNA encodingsaid segment; iv) is an expression vector; v) further comprises anorigin of replication; vi) is from a natural source; vii) comprises adetectable label; viii) comprises synthetic nucleotide sequence; ix) isless than 6 kb, preferably less than 3 kb; x) is from a primate; xi)comprises a natural full length coding sequence; xii) is a hybridizationprobe for a gene encoding said DIRS1; or xiii) is a PCR primer, PCRproduct, or mutagenesis primer; or B) DIRS2 polypeptide of claim 1,wherein said: a) DIRS2 is from a human; or b) said nucleic acid: i)encodes an antigenic peptide sequence of Table 2; ii) encodes aplurality of antigenic peptide sequences of Table 2; iii) exhibitsidentity over at least 30 nucleotides to a natural cDNA encoding saidsegment; iv) is an expression vector; v) further comprises an origin ofreplication; vi) is from a natural source; vii) comprises a detectablelabel; viii) comprises synthetic nucleotide sequence; ix) is less than 6kb, preferably less than 3 kb; x) is from a primate; xi) comprises anatural full length coding sequence; xii) is a hybridization probe for agene encoding said DIRS2; or xiii) is a PCR primer, PCR product, ormutagenesis primer.
 13. A cell or tissue comprising said recombinantnucleic acid of claim
 12. 14. The cell of claim 13, wherein said cellis: a) a prokaryotic cell; b) a eukaryotic cell; c) a bacterial cell; d)a yeast cell; e) an insect cell; f) a mammalian cell; g) a mouse cell;h) a primate cell; or i) a human cell.
 15. A kit comprising said nucleicacid of claim 12, and: a) a compartment comprising said nucleic acid; b)a compartment further comprising a primate DIRS1 polypeptide; c) acompartment further comprising a primate DIRS2 polypeptide; or d)instructions for use or disposal of reagents in said kit.
 16. A nucleicacid which: a) hybridizes under wash conditions of 30 minutes at 30° C.and less than 2M salt to the coding portion of SEQ ID NO: 1; b)hybridizes under wash conditions of 30 minutes at 30° C. and less than2M salt to the coding portion of SEQ ID NO: 3; c) exhibits identity overa stretch of at least about 30 nucleotides to a primate DIRS1; or d)exhibits identity over a stretch of at least about 30 nucleotides to aprimate DIRS2.
 17. The nucleic acid of claim 16, wherein: a) said washconditions are at 45° C. and/or 500 mM salt; or b) said stretch is atleast 55 nucleotides.
 18. The nucleic acid of claim 16, wherein: a) saidwash conditions are at 55° C. and/or 150 mM salt; or b) said stretch isat least 75 nucleotides.
 19. A method of modulating physiology ordevelopment of a cell or tissue culture cells comprising contacting saidcell with an agonist or antagonist of a mammalian DIRS1 or DIRS2. 20.The method of claim 19, wherein said cell is transformed with a nucleicacid encoding a DIRS1 or DIRS2 and another cytokine receptor subunit.